Isolated human kinase proteins, nucleic acid molecules encoding human kinase proteins, and uses thereof

ABSTRACT

The present invention provides amino acid sequences of peptides that are encoded by genes within the human genome, the kinase peptides of the present invention. The present invention specifically provides isolated peptide and nucleic acid molecules, methods of identifying orthologs and paralogs of the kinase peptides, and methods of identifying modulators of the kinase peptides.

FIELD OF THE INVENTION

[0001] The present invention is in the field of kinase proteins that arerelated to the serine/threonine protein kinase subfamily, recombinantDNA molecules, and protein production. The present inventionspecifically provides novel peptides and proteins that effect proteinphosphorylation and nucleic acid molecules encoding such peptide andprotein molecules, all of which are useful in the development of humantherapeutics and diagnostic compositions and methods.

BACKGROUND OF THE INVENTION Protein Kinases

[0002] Kinases regulate many different cell proliferation,differentiation, and signaling processes by adding phosphate groups toproteins. Uncontrolled signaling has been implicated in a variety ofdisease conditions including inflammation, cancer, arteriosclerosis, andpsoriasis. Reversible protein phosphorylation is the main strategy forcontrolling activities of eukaryotic cells. It is estimated that morethan 1000 of the 10,000 proteins active in a typical mammalian cell arephosphorylated. The high energy phosphate, which drives activation, isgenerally transferred from adenosine triphosphate molecules (ATP) to aparticular protein by protein kinases and removed from that protein byprotein phosphatases. Phosphorylation occurs in response toextracellular signals (hormones, neurotransmitters, growth anddifferentiation factors, etc), cell cycle checkpoints, and environmentalor nutritional stresses and is roughly analogous to turning on amolecular switch. When the switch goes on, the appropriate proteinkinase activates a metabolic enzyme, regulatory protein, receptor,cytoskeletal protein, ion channel or pump, or transcription factor.

[0003] The kinases comprise the largest known protein group, asuperfamily of enzymes with widely varied functions and specificities.They are usually named after their substrate, their regulatorymolecules, or some aspect of a mutant phenotype. With regard tosubstrates, the protein kinases may be roughly divided into two groups;those that phosphorylate tyrosine residues (protein tyrosine kinases,PTK) and those that phosphorylate serine or threonine residues(serine/threonine kinases, STK). A few protein kinases have dualspecificity and phosphorylate threonine and tyrosine residues. Almostall kinases contain a similar 250-300 amino acid catalytic domain. TheN-terminal domain, which contains subdomains I-IV, generally folds intoa two-lobed structure, which binds and orients the ATP (or GTP) donormolecule. The larger C terminal lobe, which contains subdomains VI A-XI,binds the protein substrate and carries out the transfer of the gammaphosphate from ATP to the hydroxyl group of a serine, threonine, ortyrosine residue. Subdomain V spans the two lobes.

[0004] The kinases may be categorized into families by the differentamino acid sequences (generally between 5 and 100 residues) located oneither side of, or inserted into loops of, the kinase domain. Theseadded amino acid sequences allow the regulation of each kinase as itrecognizes and interacts with its target protein. The primary structureof the kinase domains is conserved and can be further subdivided into 11subdomains. Each of the 11 subdomains contains specific residues andmotifs or patterns of amino acids that are characteristic of thatsubdomain and are highly conserved (Hardie, G. and Hanks, S. (1995) TheProtein Kinase Facts Books, Vol I:7-20 Academic Press, San Diego,Calif.).

[0005] The second messenger dependent protein kinases primarily mediatethe effects of second messengers such as cyclic AMP (cAMP), cyclic GMP,inositol triphosphate, phosphatidylinositol, 3,4,5-triphosphate,cyclic-ADPribose, arachidonic acid, diacylglycerol andcalcium-calmodulin. The cyclic-AMP dependent protein kinases (PKA) areimportant members of the STK family. Cyclic-AMP is an intracellularmediator of hormone action in all prokaryotic and animal cells that havebeen studied. Such hormone-induced cellular responses include thyroidhormone secretion, cortisol secretion, progesterone secretion, glycogenbreakdown, bone resorption, and regulation of heart rate and force ofheart muscle contraction. PKA is found in all animal cells and isthought to account for the effects of cyclic-AMP in most of these cells.Altered PKA expression is implicated in a variety of disorders anddiseases including cancer, thyroid disorders, diabetes, atherosclerosis,and cardiovascular disease (Isselbacher, K. J. et al. (1994) Harrison'sPrinciples of Internal Medicine, McGraw-Hill, New York, N.Y., pp.416-431, 1887).

[0006] Calcium-calmodulin (CaM) dependent protein kinases are alsomembers of STK family. Calmodulin is a calcium receptor that mediatesmany calcium regulated processes by binding to target proteins inresponse to the binding of calcium. The principle target protein inthese processes is CaM dependent protein kinases. CaM-kinases areinvolved in regulation of smooth muscle contraction (MLC kinase),glycogen breakdown (phosphorylase kinase), and neurotransmission (CaMkinase I and CaM kinase II). CaM kinase I phosphorylates a variety ofsubstrates including the neurotransmitter related proteins synapsin Iand II, the gene transcription regulator, CREB, and the cystic fibrosisconductance regulator protein, CFTR (Haribabu, B. et al. (1995) EMBOJournal 14:3679-86). CaM II kinase also phosphorylates synapsin atdifferent sites, and controls the synthesis of catecholamines in thebrain through phosphorylation and activation of tyrosine hydroxylase.Many of the CaM kinases are activated by phosphorylation in addition tobinding to CaM. The kinase may autophosphorylate itself, or bephosphorylated by another kinase as part of a “kinase cascade”.

[0007] Another ligand-activated protein kinase is 5′-AMP-activatedprotein kinase (AMPK) (Gao, G. et al. (1996) J. Biol Chem. 15:8675-81).Mammalian AMPK is a regulator of fatty acid and sterol synthesis throughphosphorylation of the enzymes acetyl-CoA carboxylase andhydroxymethylglutaryl-CoA reductase and mediates responses of thesepathways to cellular stresses such as heat shock and depletion ofglucose and ATP. AMPK is a heterotrimeric complex comprised of acatalytic alpha subunit and two non-catalytic beta and gamma subunitsthat are believed to regulate the activity of the alpha subunit.Subunits of AMPK have a much wider distribution in non-lipogenic tissuessuch as brain, heart, spleen, and lung than expected. This distributionsuggests that its role may extend beyond regulation of lipid metabolismalone.

[0008] The mitogen-activated protein kinases (MAP) are also members ofthe STK family. MAP kinases also regulate intracellular signalingpathways. They mediate signal transduction from the cell surface to thenucleus via phosphorylation cascades. Several subgroups have beenidentified, and each manifests different substrate specificities andresponds to distinct extracellular stimuli (Egan, S. E. and Weinberg, R.A. (1993) Nature 365:781-783). MAP kinase signaling pathways are presentin mammalian cells as well as in yeast. The extracellular stimuli thatactivate mammalian pathways include epidermal growth factor (EGF),ultraviolet light, hyperosmolar medium, heat shock, endotoxiclipopolysaccharide (LPS), and pro-inflammatory cytokines such as tumornecrosis factor (TNF) and interleukin-1 (IL-1).

[0009] PRK (proliferation-related kinase) is a serum/cytokine inducibleSTK that is involved in regulation of the cell cycle and cellproliferation in human megakaroytic cells (Li, B. et al. (1996) J. Biol.Chem. 271:19402-8). PRK is related to the polo (derived from humans pologene) family of STKs implicated in cell division. PRK is downregulatedin lung tumor tissue and may be a proto-oncogene whose deregulatedexpression in normal tissue leads to oncogenic transformation. AlteredMAP kinase expression is implicated in a variety of disease conditionsincluding cancer, inflammation, immune disorders, and disordersaffecting growth and development.

[0010] The cyclin-dependent protein kinases (CDKs) are another group ofSTKs that control the progression of cells through the cell cycle.Cyclins are small regulatory proteins that act by binding to andactivating CDKs that then trigger various phases of the cell cycle byphosphorylating and activating selected proteins involved in the mitoticprocess. CDKs are unique in that they require multiple inputs to becomeactivated. In addition to the binding of cyclin, CDK activation requiresthe phosphorylation of a specific threonine residue and thedephosphorylation of a specific tyrosine residue.

[0011] Protein tyrosine kinases, PTKs, specifically phosphorylatetyrosine residues on their target proteins and may be divided intotransmembrane, receptor PTKs and nontransmembrane, non-receptor PTKs.Transmembrane protein-tyrosine kinases are receptors for most growthfactors. Binding of growth factor to the receptor activates the transferof a phosphate group from ATP to selected tyrosine side chains of thereceptor and other specific proteins. Growth factors (GF) associatedwith receptor PTKs include; epidermal GF, platelet-derived GF,fibroblast GF, hepatocyte GF, insulin and insulin-like GFs, nerve GF,vascular endothelial GF, and macrophage colony stimulating factor.

[0012] Non-receptor PTKs lack transmembrane regions and, instead, formcomplexes with the intracellular regions of cell surface receptors. Suchreceptors that function through non-receptor PTKs include those forcytokines, hormones (growth hormone and prolactin) and antigen-specificreceptors on T and B lymphocytes.

[0013] Many of these PTKs were first identified as the products ofmutant oncogenes in cancer cells where their activation was no longersubject to normal cellular controls. In fact, about one third of theknown oncogenes encode PTKs, and it is well known that cellulartransformation (oncogenesis) is often accompanied by increased tyrosinephosphorylation activity (Carbonneau H and Tonks N K (1992) Annu. Rev.Cell. Biol. 8:463-93). Regulation of PTK activity may therefore be animportant strategy in controlling some types of cancer.

Serine/Threonine Protein Kinases, Ellis-van Creveld Syndrome (EvC) andWevers Acrodental Dysostosis

[0014] The novel human protein, and encoding gene, provided by thepresent invention is related to the family of serine/threonine proteinkinases. The gene provided by the present invention shows a particularlyhigh degree of similarity to the EVC gene, which is associated withEllis-van Creveld syndrome (EvC) and Weyers acrodental dysostosis(Ruiz-Perez et al., Nat Genet Mar. 24 , 2000;(3):283-6). The EVC geneencodes a 992 amino acid protein that is mutated in EvC and Weyersacrodental dysostosis. EvC and Weyers acrodental dysostosis may both beallelic conditions (Ruiz-Perez et al., Nat Genet Mar. 24,2000(3):283-6). EvC is an autosomal recessive skeletal dysplasia.Individuals with EvC generally have short limbs and short ribs,postaxial polydactyly, dysplastic nails and teeth, and, commonly,congenital cardiac defects. Weyers acrodental dysostosis is an autosomaldominant disorder characterized by features that are similar to, butmilder than, EvC (Ruiz-Perez et al., Nat Genet Mar. 24, 2000(3):283-6).

[0015] The EVC gene overlaps the collapsin response mediator protein-1(CRMP1) gene in the 3′ UTR region and a portion of the coding region(Ruiz-Perez et al., Nat Genet Mar. 24, 2000(3):283-6), suggesting afunctional correlation. For a further review of CRMP1, see Hamajima etal., Gene 180: 157-163, 1996.

[0016] For a further review of EvC and Weyers acrodental dysostosis, seeDigilio et al., Hum. Genet. 96: 251-253, 1996; McKusick, Nature Genet.24: 203-204, 2000; McKusick et al., Bull. Johns Hopkins Hosp. 115:306-336, 1964; Spranger et al., Clin. Genet. 47: 217-220, 1995; Roubiceket al., Clin. Genet. 26: 587-590, 1984; and Howard et al., Am. J. Hum.Genet. 61: 1405-1412, 1997.

[0017] Kinase proteins, particularly members of the serine/threonineprotein kinase subfamily, are a major target for drug action anddevelopment. Accordingly, it is valuable to the field of pharmaceuticaldevelopment to identify and characterize previously unknown members ofthis subfamily of kinase proteins. The present invention advances thestate of the art by providing previously unidentified human kinaseproteins that have homology to members of the serine/threonine proteinkinase subfamily.

SUMMARY OF THE INVENTION

[0018] The present invention is based in part on the identification ofamino acid sequences of human kinase peptides and proteins that arerelated to the serine/threonine protein kinase subfamily, as well asallelic variants and other mammalian orthologs thereof. These uniquepeptide sequences, and nucleic acid sequences that encode thesepeptides, can be used as models for the development of human therapeutictargets, aid in the identification of therapeutic proteins, and serve astargets for the development of human therapeutic agents that modulatekinase activity in cells and tissues that express the kinase.Experimental data as provided in FIG. 1 indicates expression in humansin prostate, lung, and whole brain.

DESCRIPTION OF THE FIGURE SHEETS

[0019]FIG. 1 provides the nucleotide sequence of a cDNA molecule ortranscript sequence that encodes the kinase protein of the presentinvention. (SEQ ID NO: 1) In addition, structure and functionalinformation is provided, such as ATG start, stop and tissuedistribution, where available, that allows one to readily determinespecific uses of inventions based on this molecular sequence.Experimental data as provided in FIG. 1 indicates expression in humansin prostate, lung, and whole brain.

[0020]FIG. 2 provides the predicted amino acid sequence of the kinase ofthe present invention. (SEQ ID NO: 2) In addition structure andfunctional information such as protein family, function, andmodification sites is provided where available, allowing one to readilydetermine specific uses of inventions based on this molecular sequence.

[0021]FIG. 3 provides genomic sequences that span the gene encoding thekinase protein of the present invention. (SEQ ID NO: 3) In additionstructure and functional information, such as intron/exon structure,promoter location, etc., is provided where available, allowing one toreadily determine specific uses of inventions based on this molecularsequence. As indicated by the data presented inAs illustrated in FIG. 3,SNPs were identified at 17 different nucleotide positions.

DETAILED DESCRIPTION OF THE INVENTION General Description

[0022] The present invention is based on the sequencing of the humangenome. During the sequencing and assembly of the human genome, analysisof the sequence information revealed previously unidentified fragmentsof the human genome that encode peptides that share structural and/orsequence homology to protein/peptide/domains identified andcharacterized within the art as being a kinase protein or part of akinase protein and are related to the serine/threonine protein kinasesubfamily. Utilizing these sequences, additional genomic sequences wereassembled and transcript and/or cDNA sequences were isolated andcharacterized. Based on this analysis, the present invention providesamino acid sequences of human kinase peptides and proteins that arerelated to the serine/threonine protein kinase subfamily, nucleic acidsequences in the form of transcript sequences, cDNA sequences and/orgenomic sequences that encode these kinase peptides and proteins,nucleic acid variation (allelic information), tissue distribution ofexpression, and information about the closest art knownprotein/peptide/domain that has structural or sequence homology to thekinase of the present invention.

[0023] In addition to being previously unknown, the peptides that areprovided in the present invention are selected based on their ability tobe used for the development of commercially important products andservices. Specifically, the present peptides are selected based onhomology and/or structural relatedness to known kinase proteins of theserine/threonine protein kinase subfamily and the expression patternobserved. Experimental data as provided in FIG. 1 indicates expressionin humans in prostate, lung, and whole brain. The art has clearlyestablished the commercial importance of members of this family ofproteins and proteins that have expression patterns similar to that ofthe present gene. Some of the more specific features of the peptides ofthe present invention, and the uses thereof, are described herein,particularly in the Background of the Invention and in the annotationprovided in the Figures, and/or are known within the art for each of theknown serine/threonine protein family or subfamily of kinase proteins.

Specific Embodiments Peptide Molecules

[0024] The present invention provides nucleic acid sequences that encodeprotein molecules that have been identified as being members of thekinase family of proteins and are related to the serine/threonineprotein kinase subfamily (protein sequences are provided in FIG. 2,transcript/cDNA sequences are provided in FIG. 1 and genomic sequencesare provided in FIG. 3). The peptide sequences provided in FIG. 2, aswell as the obvious variants described herein, particularly allelicvariants as identified herein and using the information in FIG. 3, willbe referred herein as the kinase peptides of the present invention,kinase peptides, or peptides/proteins of the present invention.

[0025] The present invention provides isolated peptide and proteinmolecules that consist of, consist essentially of, or comprise the aminoacid sequences of the kinase peptides disclosed in the FIG. 2, (encodedby the nucleic acid molecule shown in FIG. 1, transcript/cDNA or FIG. 3,genomic sequence), as well as all obvious variants of these peptidesthat are within the art to make and use. Some of these variants aredescribed in detail below.

[0026] As used herein, a peptide is said to be “isolated” or “purified”when it is substantially free of cellular material or free of chemicalprecursors or other chemicals. The peptides of the present invention canbe purified to homogeneity or other degrees of purity. The level ofpurification will be based on the intended use. The critical feature isthat the preparation allows for the desired function of the peptide,even if in the presence of considerable amounts of other components (thefeatures of an isolated nucleic acid molecule is discussed below).

[0027] In some uses, “substantially free of cellular material” includespreparations of the peptide having less than about 30% (by dry weight)other proteins (i.e., contaminating protein), less than about 20% otherproteins, less than about 10% other proteins, or less than about 5%other proteins. When the peptide is recombinantly produced, it can alsobe substantially free of culture medium, i.e., culture medium representsless than about 20% of the volume of the protein preparation.

[0028] The language “substantially free of chemical precursors or otherchemicals” includes preparations of the peptide in which it is separatedfrom chemical precursors or other chemicals that are involved in itssynthesis. In one embodiment, the language “substantially free ofchemical precursors or other chemicals” includes preparations of thekinase peptide having less than about 30% (by dry weight) chemicalprecursors or other chemicals, less than about 20% chemical precursorsor other chemicals, less than about 10% chemical precursors or otherchemicals, or less than about 5% chemical precursors or other chemicals.

[0029] The isolated kinase peptide can be purified from cells thatnaturally express it, purified from cells that have been altered toexpress it (recombinant), or synthesized using known protein synthesismethods. Experimental data as provided in FIG. 1 indicates expression inhumans in prostate, lung, and whole brain. For example, a nucleic acidmolecule encoding the kinase peptide is cloned into an expressionvector, the expression vector introduced into a host cell and theprotein expressed in the host cell. The protein can then be isolatedfrom the cells by an appropriate purification scheme using standardprotein purification techniques. Many of these techniques are describedin detail below.

[0030] Accordingly, the present invention provides proteins that consistof the amino acid sequences provided in FIG. 2 (SEQ ID NO: 2), forexample, proteins encoded by the transcript/cDNA nucleic acid sequencesshown in FIG. 1 (SEQ ID NO: 1) and the genomic sequences provided inFIG. 3 (SEQ ID NO: 3). The amino acid sequence of such a protein isprovided in FIG. 2. A protein consists of an amino acid sequence whenthe amino acid sequence is the final amino acid sequence of the protein.

[0031] The present invention further provides proteins that consistessentially of the amino acid sequences provided in FIG. 2 (SEQ ID NO:2), for example, proteins encoded by the transcript/cDNA nucleic acidsequences shown in FIG. 1 (SEQ ID NO: 1) and the genomic sequencesprovided in FIG. 3 (SEQ ID NO: 3). A protein consists essentially of anamino acid sequence when such an amino acid sequence is present withonly a few additional amino acid residues, for example from about 1 toabout 100 or so additional residues, typically from 1 to about 20additional residues in the final protein.

[0032] The present invention further provides proteins that comprise theamino acid sequences provided in FIG. 2 (SEQ ID NO: 2), for example,proteins encoded by the transcript/cDNA nucleic acid sequences shown inFIG. 1 (SEQ ID NO: 1) and the genomic sequences provided in FIG. 3 (SEQID NO: 3). A protein comprises an amino acid sequence when the aminoacid sequence is at least part of the final amino acid sequence of theprotein. In such a fashion, the protein can be only the peptide or haveadditional amino acid molecules, such as amino acid residues (contiguousencoded sequence) that are naturally associated with it or heterologousamino acid residues/peptide sequences. Such a protein can have a fewadditional amino acid residues or can comprise several hundred or moreadditional amino acids. The preferred classes of proteins that arecomprised of the kinase peptides of the present invention are thenaturally occurring mature proteins. A brief description of how varioustypes of these proteins can be made/isolated is provided below.

[0033] The kinase peptides of the present invention can be attached toheterologous sequences to form chimeric or fusion proteins. Suchchimeric and fusion proteins comprise a kinase peptide operativelylinked to a heterologous protein having an amino acid sequence notsubstantially homologous to the kinase peptide. “Operatively linked”indicates that the kinase peptide and the heterologous protein are fusedin-frame. The heterologous protein can be fused to the N-tenninus orC-terminus of the kinase peptide.

[0034] In some uses, the fusion protein does not affect the activity ofthe kinase peptide per se. For example, the fusion protein can include,but is not limited to, enzymatic fusion proteins, for examplebeta-galactosidase fusions, yeast two-hybrid GAL fusions, poly-Hisfusions, MYC-tagged, HI-tagged and Ig fusions. Such fusion proteins,particularly poly-His fusions, can facilitate the purification ofrecombinant kinase peptide. In certain host cells (e.g., mammalian hostcells), expression and/or secretion of a protein can be increased byusing a heterologous signal sequence.

[0035] A chimeric or fusion protein can be produced by standardrecombinant DNA techniques. For example, DNA fragments coding for thedifferent protein sequences are ligated together in-frame in accordancewith conventional techniques. In another embodiment, the fusion gene canbe synthesized by conventional techniques including automated DNAsynthesizers. Alternatively, PCR amplification of gene fragments can becarried out using anchor primers which give rise to complementaryoverhangs between two consecutive gene fragments which can subsequentlybe annealed and re-amplified to generate a chimeric gene sequence (seeAusubel et al., Current Protocols in Molecular Biology, 1992). Moreover,many expression vectors are commercially available that already encode afusion moiety (e.g., a GST protein). A kinase peptide-encoding nucleicacid can be cloned into such an expression vector such that the fusionmoiety is linked in-frame to the kinase peptide.

[0036] As mentioned above, the present invention also provides andenables obvious variants of the amino acid sequence of the proteins ofthe present invention, such as naturally occurring mature forms of thepeptide, allelic/sequence variants of the peptides, non-naturallyoccurring recombinantly derived variants of the peptides, and orthologsand paralogs of the peptides. Such variants can readily be generatedusing art-known techniques in the fields of recombinant nucleic acidtechnology and protein biochemistry. It is understood, however, thatvariants exclude any amino acid sequences disclosed prior to theinvention.

[0037] Such variants can readily be identified/made using moleculartechniques and the sequence information disclosed herein. Further, suchvariants can readily be distinguished from other peptides based onsequence and/or structural homology to the kinase peptides of thepresent invention. The degree of homology/identity present will be basedprimarily on whether the peptide is a functional variant ornon-functional variant, the amount of divergence present in the paralogfamily and the evolutionary distance between the orthologs.

[0038] To determine the percent identity of two amino acid sequences ortwo nucleic acid sequences, the sequences are aligned for optimalcomparison purposes (e.g., gaps can be introduced in one or both of afirst and a second amino acid or nucleic acid sequence for optimalalignment and non-homologous sequences can be disregarded for comparisonpurposes). In a preferred embodiment, at least 30%, 40%, 50%, 60%, 70%,80%, or 90% or more of the length of a reference sequence is aligned forcomparison purposes. The amino acid residues or nucleotides atcorresponding amino acid positions or nucleotide positions are thencompared. When a position in the first sequence is occupied by the sameamino acid residue or nucleotide as the corresponding position in thesecond sequence, then the molecules are identical at that position (asused herein amino acid or nucleic acid “identity” is equivalent to aminoacid or nucleic acid “homology”). The percent identity between the twosequences is a function of the number of identical positions shared bythe sequences, taking into account the number of gaps, and the length ofeach gap, which need to be introduced for optimal alignment of the twosequences.

[0039] The comparison of sequences and determination of percent identityand similarity between two sequences can be accomplished using amathematical algorithm. (Computational Molecular Biology, Lesk, A. M.,ed., Oxford University Press, New York, 1988; Biocomputing: Informaticsand Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993;Computer Analysis of Sequence Data, Part 1, Griffin, A. M., and Griffin,H. G., eds., Humana Press, N.J., 1994; Sequence Analysis in MolecularBiology, von Heinje, G., Academic Press, 1987; and Sequence AnalysisPrimer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York,1991). In a preferred embodiment, the percent identity between two aminoacid sequences is determined using the Needleman and Wunsch (J. Mol.Biol. (48):444-453 (1970)) algorithm which has been incorporated intothe GAP program in the GCG software package (available athttp://www.gcg.com), using either a Blossom 62 matrix or a PAM250matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a lengthweight of 1, 2, 3, 4, 5, or 6. In yet another preferred embodiment, thepercent identity between two nucleotide sequences is determined usingthe GAP program in the GCG software package (Devereux, J., et al.,Nucleic Acids Res. 12(1):387 (1984)) (available at http://www.gcg.com),using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80and a length weight of 1, 2, 3, 4, 5, or 6. In another embodiment, thepercent identity between two amino acid or nucleotide sequences isdetermined using the algorithm of E. Myers and W. Miller (CABIOS,4:11-17 (1989)) which has been incorporated into the ALIGN program(version 2.0), using a PAM120 weight residue table, a gap length penaltyof 12 and a gap penalty of 4.

[0040] The nucleic acid and protein sequences of the present inventioncan further be used as a “query sequence” to perform a search againstsequence databases to, for example, identify other family members orrelated sequences. Such searches can be performed using the NBLAST andXBLAST programs (version 2.0) of Altschul, et al. (J. Mol. Biol.215:403-10 (1990)). BLAST nucleotide searches can be performed with theNBLAST program, score=100, wordlength=12 to obtain nucleotide sequenceshomologous to the nucleic acid molecules of the invention. BLAST proteinsearches can be performed with the XBLAST program, score=50,wordlength=3 to obtain amino acid sequences homologous to the proteinsof the invention. To obtain gapped alignments for comparison purposes,Gapped BLAST can be utilized as described in Altschul et al. (NucleicAcids Res. 25(17):3389-3402 (1997)). When utilizing BLAST and gappedBLAST programs, the default parameters of the respective programs (e.g.,XBLAST and NBLAST) can be used.

[0041] Full-length pre-processed forms, as well as mature processedforms, of proteins that comprise one of the peptides of the presentinvention can readily be identified as having complete sequence identityto one of the kinase peptides of the present invention as well as beingencoded by the same genetic locus as the kinase peptide provided herein.As indicated by the data presented in FIG. 3, the map position wasdetermined to be on chromosome 10 by ePCR.

[0042] Allelic variants of a kinase peptide can readily be identified asbeing a human protein having a high degree (significant) of sequencehomology/identity to at least a portion of the kinase peptide as well asbeing encoded by the same genetic locus as the kinase peptide providedherein. Genetic locus can readily be determined based on the genomicinformation provided in FIG. 3, such as the genomic sequence mapped tothe reference human. As indicated by the data presented in FIG. 3, themap position was determined to be on chromosome 10 by ePCR. As usedherein, two proteins (or a region of the proteins) have significanthomology when the amino acid sequences are typically at least about70-80%, 80-90%, and more typically at least about 90-95% or morehomologous. A significantly homologous amino acid sequence, according tothe present invention, will be encoded by a nucleic acid sequence thatwill hybridize to a kinase peptide encoding nucleic acid molecule understringent conditions as more fully described below.

[0043]FIG. 3 provides information on SNPs that have been found in thegene encoding the kinase protein of the present invention. SNPs wereidentified at 17 different nucleotide positions. SNPs such as these,particularly SNPs outside the ORF and in the first intron, may affectcontrol/regulatory elements.

[0044] Paralogs of a kinase peptide can readily be identified as havingsome degree of significant sequence homology/identity to at least aportion of the kinase peptide, as being encoded by a gene from humans,and as having similar activity or function. Two proteins will typicallybe considered paralogs when the amino acid sequences are typically atleast about 60% or greater, and more typically at least about 70% orgreater homology through a given region or domain. Such paralogs will beencoded by a nucleic acid sequence that will hybridize to a kinasepeptide encoding nucleic acid molecule under moderate to stringentconditions as more fully described below.

[0045] Orthologs of a kinase peptide can readily be identified as havingsome degree of significant sequence homology/identity to at least aportion of the kinase peptide as well as being encoded by a gene fromanother organism. Preferred orthologs will be isolated from mammals,preferably primates, for the development of human therapeutic targetsand agents. Such orthologs will be encoded by a nucleic acid sequencethat will hybridize to a kinase peptide encoding nucleic acid moleculeunder moderate to stringent conditions, as more fully described below,depending on the degree of relatedness of the two organisms yielding theproteins.

[0046] Non-naturally occurring variants of the kinase peptides of thepresent invention can readily be generated using recombinant techniques.Such variants include, but are not limited to deletions, additions andsubstitutions in the amino acid sequence of the kinase peptide. Forexample, one class of substitutions are conserved amino acidsubstitution. Such substitutions are those that substitute a given aminoacid in a kinase peptide by another amino acid of like characteristics.Typically seen as conservative substitutions are the replacements, onefor another, among the aliphatic amino acids Ala, Val, Leu, and Ile;interchange of the hydroxyl residues Ser and Thr, exchange of the acidicresidues Asp and Glu; substitution between the amide residues Asn andGln; exchange of the basic residues Lys and Arg; and replacements amongthe aromatic residues Phe and Tyr. Guidance concerning which amino acidchanges are likely to be phenotypically silent are found in Bowie etal., Science 247:1306-1310 (1990).

[0047] Variant kinase peptides can be fully functional or can lackfunction in one or more activities, e.g. ability to bind substrate,ability to phosphorylate substrate, ability to mediate signaling, etc.Fully functional variants typically contain only conservative variationor variation in non-critical residues or in non-critical regions. FIG. 2provides the result of protein analysis and can be used to identifycritical domains/regions. Functional variants can also containsubstitution of similar amino acids that result in no change or aninsignificant change in function. Alternatively, such substitutions maypositively or negatively affect function to some degree.

[0048] Non-functional variants typically contain one or morenon-conservative amino acid substitutions, deletions, insertions,inversions, or truncation or a substitution, insertion, inversion, ordeletion in a critical residue or critical region.

[0049] Amino acids that are essential for function can be identified bymethods known in the art, such as site-directed mutagenesis oralanine-scanning mutagenesis (Cunningham et al., Science 244:1081-1085(1989)), particularly using the results provided in FIG. 2. The latterprocedure introduces single alanine mutations at every residue in themolecule. The resulting mutant molecules are then tested for biologicalactivity such as kinase activity or in assays such as an in vitroproliferative activity. Sites that are critical for bindingpartner/substrate binding can also be determined by structural analysissuch as crystallization, nuclear magnetic resonance or photoaffinitylabeling (Smith et al., J. Mol. Biol. 224:899-904 (1992); de Vos et al.Science 255:306-312 (1992)).

[0050] The present invention further provides fragments of the kinasepeptides, in addition to proteins and peptides that comprise and consistof such fragments, particularly those comprising the residues identifiedin FIG. 2. The fragments to which the invention pertains, however, arenot to be construed as encompassing fragments that may be disclosedpublicly prior to the present invention.

[0051] As used herein, a fragment comprises at least 8, 10, 12, 14, 16,or more contiguous amino acid residues from a kinase peptide. Suchfragments can be chosen based on the ability to retain one or more ofthe biological activities of the kinase peptide or could be chosen forthe ability to perform a function, e.g. bind a substrate or act as animmunogen. Particularly important fragments are biologically activefragments, peptides that are, for example, about 8 or more amino acidsin length. Such fragments will typically comprise a domain or motif ofthe kinase peptide, e.g., active site, a transmembrane domain or asubstrate-binding domain. Further, possible fragments include, but arenot limited to, domain or motif containing fragments, soluble peptidefragments, and fragments containing immunogenic structures. Predicteddomains and functional sites are readily identifiable by computerprograms well known and readily available to those of skill in the art(e.g., PROSITE analysis). The results of one such analysis are providedin FIG. 2.

[0052] Polypeptides often contain amino acids other than the 20 aminoacids commonly referred to as the 20 naturally occurring amino acids.Further, many amino acids, including the terminal amino acids, may bemodified by natural processes, such as processing and otherpost-translational modifications, or by chemical modification techniqueswell known in the art. Common modifications that occur naturally inkinase peptides are described in basic texts, detailed monographs, andthe research literature, and they are well known to those of skill inthe art (some of these features are identified in FIG. 2).

[0053] Known modifications include, but are not limited to, acetylation,acylation, ADP-ribosylation, amidation, covalent attachment of flavin,covalent attachment of a heme moiety, covalent attachment of anucleotide or nucleotide derivative, covalent attachment of a lipid orlipid derivative, covalent attachment of phosphotidylinositol,cross-linking, cyclization, disulfide bond formation, demethylation,formation of covalent crosslinks, formation of cystine, formation ofpyroglutamate, formylation, gamma carboxylation, glycosylation, GPIanchor formation, hydroxylation, iodination, methylation,myristoylation, oxidation, proteolytic processing, phosphorylation,prenylation, racemization, selenoylation, sulfation, transfer-RNAmediated addition of amino acids to proteins such as arginylation, andubiquitination.

[0054] Such modifications are well known to those of skill in the artand have been described in great detail in the scientific literature.Several particularly common modifications, glycosylation, lipidattachment, sulfation, gamma-carboxylation of glutamic acid residues,hydroxylation and ADP-ribosylation, for instance, are described in mostbasic texts, such as Proteins—Structure and Molecular Properties, 2ndEd., T. E. Creighton, W. H. Freeman and Company, New York (1993). Manydetailed reviews are available on this subject, such as by Wold, F.,Posttranslational Covalent Modification of Proteins, B. C. Johnson, Ed.,Academic Press, New York 1-12 (1983); Seifter et al. (Meth. Enzymol.182: 626-646 (1990)) and Rattan et al. (Ann. N.Y Acad. Sci. 663:48-62(1992)).

[0055] Accordingly, the kinase peptides of the present invention alsoencompass derivatives or analogs in which a substituted amino acidresidue is not one encoded by the genetic code, in which a substituentgroup is included, in which the mature kinase peptide is fused withanother compound, such as a compound to increase the half-life of thekinase peptide (for example, polyethylene glycol), or in which theadditional amino acids are fused to the mature kinase peptide, such as aleader or secretory sequence or a sequence for purification of themature kinase peptide or a pro-protein sequence.

Protein/Peptide Uses

[0056] The proteins of the present invention can be used in substantialand specific assays related to the functional information provided inthe Figures; to raise antibodies or to elicit another immune response;as a reagent (including the labeled reagent) in assays designed toquantitatively determine levels of the protein (or its binding partneror ligand) in biological fluids; and as markers for tissues in which thecorresponding protein is preferentially expressed (either constitutivelyor at a particular stage of tissue differentiation or development or ina disease state). Where the protein binds or potentially binds toanother protein or ligand (such as, for example, in a kinase-effectorprotein interaction or kinase-ligand interaction), the protein can beused to identify the binding partner/ligand so as to develop a system toidentify inhibitors of the binding interaction. Any or all of these usesare capable of being developed into reagent grade or kit format forcommercialization as commercial products.

[0057] Methods for performing the uses listed above are well known tothose skilled in the art. References disclosing such methods include“Molecular Cloning: A Laboratory Manual”, 2d ed., Cold Spring HarborLaboratory Press, Sambrook, J., E. F. Fritsch and T. Maniatis eds.,1989, and “Methods in Enzymology: Guide to Molecular CloningTechniques”, Academic Press, Berger, S. L. and A. R. Kimmel eds., 1987.

[0058] The potential uses of the peptides of the present invention arebased primarily on the source of the protein as well as the class/actionof the protein. For example, kinases isolated from humans and theirhuman/mammalian orthologs serve as targets for identifying agents foruse in mammalian therapeutic applications, e.g. a human drug,particularly in modulating a biological or pathological response in acell or tissue that expresses the kinase. Experimental data as providedin FIG. 1 indicates that kinase proteins of the present invention areexpressed in humans in prostate, lung, and whole brain, as indicated byvirtual northern blot analysis. In addition, PCR-based tissue screeningpanel indicates expression in whole brain. A large percentage ofpharmaceutical agents are being developed that modulate the activity ofkinase proteins, particularly members of the serine/threonine proteinsubfamily (see Background of the Invention). The structural andfunctional information provided in the Background and Figures providespecific and substantial uses for the molecules of the presentinvention, particularly in combination with the expression informationprovided in FIG. 1. Experimental data as provided in FIG. 1 indicatesexpression in humans in prostate, lung, and whole brain. Such uses canreadily be determined using the information provided herein, that whichis known in the art, and routine experimentation.

[0059] The proteins of the present invention (including variants andfragments that may have been disclosed prior to the present invention)are useful for biological assays related to kinases that are related tomembers of the serine/threonine protein subfamily. Such assays involveany of the known kinase functions or activities or properties useful fordiagnosis and treatment of kinase-related conditions that are specificfor the subfamily of kinases that the one of the present inventionbelongs to, particularly in cells and tissues that express the kinase.Experimental data as provided in FIG. 1 indicates that kinase proteinsof the present invention are expressed in humans in prostate, lung, andwhole brain, as indicated by virtual northern blot analysis. Inaddition, PCR-based tissue screening panel indicates expression in wholebrain.

[0060] The proteins of the present invention are also useful in drugscreening assays, in cell-based or cell-free systems. Cell-based systemscan be native, i.e., cells that normally express the kinase, as a biopsyor expanded in cell culture. Experimental data as provided in FIG. 1indicates expression in humans in prostate, lung, and whole brain. In analternate embodiment, cell-based assays involve recombinant host cellsexpressing the kinase protein.

[0061] The polypeptides can be used to identify compounds that modulatekinase activity of the protein in its natural state or an altered formthat causes a specific disease or pathology associated with the kinase.Both the kinases of the present invention and appropriate variants andfragments can be used in high-throughput screens to assay candidatecompounds for the ability to bind to the kinase. These compounds can befurther screened against a functional kinase to determine the effect ofthe compound on the kinase activity. Further, these compounds can betested in animal or invertebrate systems to determineactivity/effectiveness. Compounds can be identified that activate(agonist) or inactivate (antagonist) the kinase to a desired degree.

[0062] Further, the proteins of the present invention can be used toscreen a compound for the ability to stimulate or inhibit interactionbetween the kinase protein and a molecule that normally interacts withthe kinase protein, e.g. a substrate or a component of the signalpathway that the kinase protein normally interacts (for example, anotherkinase). Such assays typically include the steps of combining the kinaseprotein with a candidate compound under conditions that allow the kinaseprotein, or fragment, to interact with the target molecule, and todetect the formation of a complex between the protein and the target orto detect the biochemical consequence of the interaction with the kinaseprotein and the target, such as any of the associated effects of signaltransduction such as protein phosphorylation, cAMP turnover, andadenylate cyclase activation, etc.

[0063] Candidate compounds include, for example, 1) peptides such assoluble peptides, including Ig-tailed fusion peptides and members ofrandom peptide libraries (see, e.g., Lam et al, Nature 354:82-84 (1991);Houghten et al., Nature 354:84-86 (1991)) and combinatorialchemistry-derived molecular libraries made of D- and/or L- configurationamino acids; 2) phosphopeptides (e.g., members of random and partiallydegenerate, directed phosphopeptide libraries, see, e.g., Songyang etal., Cell 72:767-778 (1993)); 3) antibodies (e.g., polyclonal,monoclonal, humanized, anti-idiotypic, chimeric, and single chainantibodies as well as Fab, F(ab′)₂, Fab expression library fragments,and epitope-binding fragments of antibodies); and 4) small organic andinorganic molecules (e.g., molecules obtained from combinatorial andnatural product libraries).

[0064] One candidate compound is a soluble fragment of the receptor thatcompetes for substrate binding. Other candidate compounds include mutantkinases or appropriate fragments containing mutations that affect kinasefunction and thus compete for substrate. Accordingly, a fragment thatcompetes for substrate, for example with a higher affinity, or afragment that binds substrate but does not allow release, is encompassedby the invention.

[0065] The invention further includes other end point assays to identifycompounds that modulate (stimulate or inhibit) kinase activity. Theassays typically involve an assay of events in the signal transductionpathway that indicate kinase activity. Thus, the phosphorylation of asubstrate, activation of a protein, a change in the expression of genesthat are up- or down-regulated in response to the kinase proteindependent signal cascade can be assayed.

[0066] Any of the biological or biochemical functions mediated by thekinase can be used as an endpoint assay. These include all of thebiochemical or biochemical/biological events described herein, in thereferences cited herein, incorporated by reference for these endpointassay targets, and other functions known to those of ordinary skill inthe art or that can be readily identified using the information providedin the Figures, particularly FIG. 2. Specifically, a biological functionof a cell or tissues that expresses the kinase can be assayed.Experimental data as provided in FIG. 1 indicates that kinase proteinsof the present invention are expressed in humans in prostate, lung, andwhole brain, as indicated by virtual northern blot analysis. Inaddition, PCR-based tissue screening panel indicates expression in wholebrain.

[0067] Binding and/or activating compounds can also be screened by usingchimeric kinase proteins in which the amino terminal extracellulardomain, or parts thereof, the entire transmembrane domain or subregions,such as any of the seven transmembrane segments or any of theintracellular or extracellular loops and the carboxy terminalintracellular domain, or parts thereof, can be replaced by heterologousdomains or subregions. For example, a substrate-binding region can beused that interacts with a different substrate then that which isrecognized by the native kinase. Accordingly, a different set of signaltransduction components is available as an end-point assay foractivation. This allows for assays to be performed in other than thespecific host cell from which the kinase is derived.

[0068] The proteins of the present invention are also useful incompetition binding assays in methods designed to discover compoundsthat interact with the kinase (e.g. binding partners and/or ligands).Thus, a compound is exposed to a kinase polypeptide under conditionsthat allow the compound to bind or to otherwise interact with thepolypeptide. Soluble kinase polypeptide is also added to the mixture. Ifthe test compound interacts with the soluble kinase polypeptide, itdecreases the amount of complex formed or activity from the kinasetarget. This type of assay is particularly useful in cases in whichcompounds are sought that interact with specific regions of the kinase.Thus, the soluble polypeptide that competes with the target kinaseregion is designed to contain peptide sequences corresponding to theregion of interest.

[0069] To perform cell free drug screening assays, it is sometimesdesirable to immobilize either the kinase protein, or fragment, or itstarget molecule to facilitate separation of complexes from uncomplexedforms of one or both of the proteins, as well as to accommodateautomation of the assay.

[0070] Techniques for immobilizing proteins on matrices can be used inthe drug screening assays. In one embodiment, a fusion protein can beprovided which adds a domain that allows the protein to be bound to amatrix. For example, glutathione-S-transferase fusion proteins can beadsorbed onto glutathione sepharose beads (Sigma Chemical, St. Louis,Mo.) or glutathione derivatized microtitre plates, which are thencombined with the cell lysates (e.g., ³⁵S-labeled) and the candidatecompound, and the mixture incubated under conditions conducive tocomplex formation (e.g., at physiological conditions for salt and pH).Following incubation, the beads are washed to remove any unbound label,and the matrix immobilized and radiolabel determined directly, or in thesupernatant after the complexes are dissociated. Alternatively, thecomplexes can be dissociated from the matrix, separated by SDS-PAGE, andthe level of kinase-binding protein found in the bead fractionquantitated from the gel using standard electrophoretic techniques. Forexample, either the polypeptide or its target molecule can beimmobilized utilizing conjugation of biotin and streptavidin usingtechniques well known in the art. Alternatively, antibodies reactivewith the protein but which do not interfere with binding of the proteinto its target molecule can be derivatized to the wells of the plate, andthe protein trapped in the wells by antibody conjugation. Preparationsof a kinase-binding protein and a candidate compound are incubated inthe kinase protein-presenting wells and the amount of complex trapped inthe well can be quantitated. Methods for detecting such complexes, inaddition to those described above for the GST-immobilized complexes,include immunodetection of complexes using antibodies reactive with thekinase protein target molecule, or which are reactive with kinaseprotein and compete with the target molecule, as well as enzyme-linkedassays which rely on detecting an enzymatic activity associated with thetarget molecule.

[0071] Agents that modulate one of the kinases of the present inventioncan be identified using one or more of the above assays, alone or incombination. It is generally preferable to use a cell-based or cell freesystem first and then confirm activity in an animal or other modelsystem. Such model systems are well known in the art and can readily beemployed in this context.

[0072] Modulators of kinase protein activity identified according tothese drug screening assays can be used to treat a subject with adisorder mediated by the kinase pathway, by treating cells or tissuesthat express the kinase. Experimental data as provided in FIG. 1indicates expression in humans in prostate, lung, and whole brain. Thesemethods of treatment include the steps of administering a modulator ofkinase activity in a pharmaceutical composition to a subject in need ofsuch treatment, the modulator being identified as described herein.

[0073] In yet another aspect of the invention, the kinase proteins canbe used as “bait proteins” in a two-hybrid assay or three-hybrid assay(see, e.g., U.S. Pat. No. 5,283,317; Zervos et al. (1993) Cell72:223-232; Madura et al. (1993) J. Biol. Chem. 268:12046-12054; Bartelet al. (1993) Biotechniques 14:920-924; Iwabuchi et al. (1993) Oncogene8:1693-1696; and Brent WO94/10300), to identify other proteins, whichbind to or interact with the kinase and are involved in kinase activity.Such kinase-binding proteins are also likely to be involved in thepropagation of signals by the kinase proteins or kinase targets as, forexample, downstream elements of a kinase-mediated signaling pathway.Alternatively, such kinase-binding proteins are likely to be kinaseinhibitors.

[0074] The two-hybrid system is based on the modular nature of mosttranscription factors, which consist of separable DNA-binding andactivation domains. Briefly, the assay utilizes two different DNAconstructs. In one construct, the gene that codes for a kinase proteinis fused to a gene encoding the DNA binding domain of a knowntranscription factor (e.g., GAL-4). In the other construct, a DNAsequence, from a library of DNA sequences, that encodes an unidentifiedprotein (“prey” or “sample”) is fused to a gene that codes for theactivation domain of the known transcription factor. If the “bait” andthe “prey” proteins are able to interact, in vivo, forming akinase-dependent complex, the DNA-binding and activation domains of thetranscription factor are brought into close proximity. This proximityallows transcription of a reporter gene (e.g., LacZ) which is operablylinked to a transcriptional regulatory site responsive to thetranscription factor. Expression of the reporter gene can be detectedand cell colonies containing the functional transcription factor can beisolated and used to obtain the cloned gene which encodes the proteinwhich interacts with the kinase protein.

[0075] This invention further pertains to novel agents identified by theabove-described screening assays. Accordingly, it is within the scope ofthis invention to further use an agent identified as described herein inan appropriate animal model. For example, an agent identified asdescribed herein (e.g., a kinase-modulating agent, an antisense kinasenucleic acid molecule, a kinase-specific antibody, or a kinase-bindingpartner) can be used in an animal or other model to determine theefficacy, toxicity, or side effects of treatment with such an agent.Alternatively, an agent identified as described herein can be used in ananimal or other model to determine the mechanism of action of such anagent. Furthermore, this invention pertains to uses of novel agentsidentified by the above-described screening assays for treatments asdescribed herein.

[0076] The kinase proteins of the present invention are also useful toprovide a target for diagnosing a disease or predisposition to diseasemediated by the peptide. Accordingly, the invention provides methods fordetecting the presence, or levels of, the protein (or encoding mRNA) ina cell, tissue, or organism. Experimental data as provided in FIG. 1indicates expression in humans in prostate, lung, and whole brain. Themethod involves contacting a biological sample with a compound capableof interacting with the kinase protein such that the interaction can bedetected. Such an assay can be provided in a single detection format ora multi-detection format such as an antibody chip array.

[0077] One agent for detecting a protein in a sample is an antibodycapable of selectively binding to protein. A biological sample includestissues, cells and biological fluids isolated from a subject, as well astissues, cells and fluids present within a subject.

[0078] The peptides of the present invention also provide targets fordiagnosing active protein activity, disease, or predisposition todisease, in a patient having a variant peptide, particularly activitiesand conditions that are known for other members of the family ofproteins to which the present one belongs. Thus, the peptide can beisolated from a biological sample and assayed for the presence of agenetic mutation that results in aberrant peptide. This includes aminoacid substitution, deletion, insertion, rearrangement, (as the result ofaberrant splicing events), and inappropriate post-translationalmodification. Analytic methods include altered electrophoretic mobility,altered tryptic peptide digest, altered kinase activity in cell-based orcell-free assay, alteration in substrate or antibody-binding pattern,altered isoelectric point, direct amino acid sequencing, and any otherof the known assay techniques useful for detecting mutations in aprotein. Such an assay can be provided in a single detection format or amulti-detection format such as an antibody chip array.

[0079] In vitro techniques for detection of peptide include enzymelinked immunosorbent assays (ELISAs), Western blots,immunoprecipitations and immunofluorescence using a detection reagent,such as an antibody or protein binding agent. Alternatively, the peptidecan be detected in vivo in a subject by introducing into the subject alabeled anti-peptide antibody or other types of detection agent. Forexample, the antibody can be labeled with a radioactive marker whosepresence and location in a subject can be detected by standard imagingtechniques. Particularly useful are methods that detect the allelicvariant of a peptide expressed in a subject and methods which detectfragments of a peptide in a sample.

[0080] The peptides are also useful in pharmacogenomic analysis.Pharmacogenomics deal with clinically significant hereditary variationsin the response to drugs due to altered drug disposition and abnormalaction in affected persons. See, e.g., Eichelbaum, M. (Clin. Exp.Phannacol. Physiol. 23(10-11):983-985 (1996)), and Linder, M. W. (Clin.Chem. 43(2):254-266 (1997)). The clinical outcomes of these variationsresult in severe toxicity of therapeutic drugs in certain individuals ortherapeutic failure of drugs in certain individuals as a result ofindividual variation in metabolism. Thus, the genotype of the individualcan determine the way a therapeutic compound acts on the body or the waythe body metabolizes the compound. Further, the activity of drugmetabolizing enzymes effects both the intensity and duration of drugaction. Thus, the pharmacogenomics of the individual permit theselection of effective compounds and effective dosages of such compoundsfor prophylactic or therapeutic treatment based on the individual'sgenotype. The discovery of genetic polymorphisms in some drugmetabolizing enzymes has explained why some patients do not obtain theexpected drug effects, show an exaggerated drug effect, or experienceserious toxicity from standard drug dosages. Polymorphisms can beexpressed in the phenotype of the extensive metabolizer and thephenotype of the poor metabolizer. Accordingly, genetic polymorphism maylead to allelic protein variants of the kinase protein in which one ormore of the kinase functions in one population is different from thosein another population. The peptides thus allow a target to ascertain agenetic predisposition that can affect treatment modality. Thus, in aligand-based treatment, polymorphism may give rise to amino terminalextracellular domains and/or other substrate-binding regions that aremore or less active in substrate binding, and kinase activation.Accordingly, substrate dosage would necessarily be modified to maximizethe therapeutic effect within a given population containing apolymorphism. As an alternative to genotyping, specific polymorphicpeptides could be identified.

[0081] The peptides are also useful for treating a disordercharacterized by an absence of, inappropriate, or unwanted expression ofthe protein. Experimental data as provided in FIG. 1 indicatesexpression in humans in prostate, lung, and whole brain. Accordingly,methods for treatment include the use of the kinase protein orfragments.

Antibodies

[0082] The invention also provides antibodies that selectively bind toone of the peptides of the present invention, a protein comprising sucha peptide, as well as variants and fragments thereof. As used herein, anantibody selectively binds a target peptide when it binds the targetpeptide and does not significantly bind to unrelated proteins. Anantibody is still considered to selectively bind a peptide even if italso binds to other proteins that are not substantially homologous withthe target peptide so long as such proteins share homology with afragment or domain of the peptide target of the antibody. In this case,it would be understood that antibody binding to the peptide is stillselective despite some degree of cross-reactivity.

[0083] As used herein, an antibody is defined in terms consistent withthat recognized within the art: they are multi-subunit proteins producedby a mammalian organism in response to an antigen challenge. Theantibodies of the present invention include polyclonal antibodies andmonoclonal antibodies, as well as fragments of such antibodies,including, but not limited to, Fab or F(ab′)₂, and Fv fragments.

[0084] Many methods are known for generating and/or identifyingantibodies to a given target peptide. Several such methods are describedby Harlow, Antibodies, Cold Spring Harbor Press, (1989).

[0085] In general, to generate antibodies, an isolated peptide is usedas an immunogen and is administered to a mammalian organism, such as arat, rabbit or mouse. The full-length protein, an antigenic peptidefragment or a fusion protein can be used. Particularly importantfragments are those covering functional domains, such as the domainsidentified in FIG. 2, and domain of sequence homology or divergenceamongst the family, such as those that can readily be identified usingprotein alignment methods and as presented in the Figures.

[0086] Antibodies are preferably prepared from regions or discretefragments of the kinase proteins. Antibodies can be prepared from anyregion of the peptide as described herein. However, preferred regionswill include those involved in function/activity and/or kinase/bindingpartner interaction. FIG. 2 can be used to identify particularlyimportant regions while sequence alignment can be used to identifyconserved and unique sequence fragments.

[0087] An antigenic fragment will typically comprise at least 8contiguous amino acid residues. The antigenic peptide can comprise,however, at least 10, 12, 14, 16 or more amino acid residues. Suchfragments can be selected on a physical property, such as fragmentscorrespond to regions that are located on the surface of the protein,e.g., hydrophilic regions or can be selected based on sequenceuniqueness (see FIG. 2).

[0088] Detection on an antibody of the present invention can befacilitated by coupling (i.e., physically linking) the antibody to adetectable substance. Examples of detectable substances include variousenzymes, prosthetic groups, fluorescent materials, luminescentmaterials, bioluminescent materials, and radioactive materials. Examplesof suitable enzymes include horseradish peroxidase, alkalinephosphatase, β-galactosidase, or acetylcholinesterase; examples ofsuitable prosthetic group complexes include streptavidin/biotin andavidin/biotin; examples of suitable fluorescent materials includeumbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine,dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; anexample of a luminescent material includes luminol; examples ofbioluminescent materials include luciferase, luciferin, and aequorin,and examples of suitable radioactive material include ¹²⁵I, ¹³¹I, ³⁵S or³H.

Antibody Uses

[0089] The antibodies can be used to isolate one of the proteins of thepresent invention by standard techniques, such as affinitychromatography or immunoprecipitation. The antibodies can facilitate thepurification of the natural protein from cells and recombinantlyproduced protein expressed in host cells. In addition, such antibodiesare useful to detect the presence of one of the proteins of the presentinvention in cells or tissues to determine the pattern of expression ofthe protein among various tissues in an organism and over the course ofnormal development. Experimental data as provided in FIG. 1 indicatesthat kinase proteins of the present invention are expressed in humans inprostate, lung, and whole brain, as indicated by virtual northern blotanalysis. In addition, PCR-based tissue screening panel indicatesexpression in whole brain. Further, such antibodies can be used todetect protein in situ, in vitro, or in a cell lysate or supernatant inorder to evaluate the abundance and pattern of expression. Also, suchantibodies can be used to assess abnormal tissue distribution orabnormal expression during development or progression of a biologicalcondition. Antibody detection of circulating fragments of the fulllength protein can be used to identify turnover.

[0090] Further, the antibodies can be used to assess expression indisease states such as in active stages of the disease or in anindividual with a predisposition toward disease related to the protein'sfunction. When a disorder is caused by an inappropriate tissuedistribution, developmental expression, level of expression of theprotein, or expressed/processed form, the antibody can be preparedagainst the normal protein. Experimental data as provided in FIG. 1indicates expression in humans in prostate, lung, and whole brain. If adisorder is characterized by a specific mutation in the protein,antibodies specific for this mutant protein can be used to assay for thepresence of the specific mutant protein.

[0091] The antibodies can also be used to assess normal and aberrantsubcellular localization of cells in the various tissues in an organism.Experimental data as provided in FIG. 1 indicates expression in humansin prostate, lung, and whole brain. The diagnostic uses can be applied,not only in genetic testing, but also in monitoring a treatmentmodality. Accordingly, where treatment is ultimately aimed at correctingexpression level or the presence of aberrant sequence and aberranttissue distribution or developmental expression, antibodies directedagainst the protein or relevant fragments can be used to monitortherapeutic efficacy.

[0092] Additionally, antibodies are useful in pharmacogenomic analysis.Thus, antibodies prepared against polymorphic proteins can be used toidentify individuals that require modified treatment modalities. Theantibodies are also useful as diagnostic tools as an immunologicalmarker for aberrant protein analyzed by electrophoretic mobility,isoelectric point, tryptic peptide digest, and other physical assaysknown to those in the art.

[0093] The antibodies are also useful for tissue typing. Experimentaldata as provided in FIG. 1 indicates expression in humans in prostate,lung, and whole brain. Thus, where a specific protein has beencorrelated with expression in a specific tissue, antibodies that arespecific for this protein can be used to identify a tissue type.

[0094] The antibodies are also useful for inhibiting protein function,for example, blocking the binding of the kinase peptide to a bindingpartner such as a substrate. These uses can also be applied in atherapeutic context in which treatment involves inhibiting the protein'sfunction. An antibody can be used, for example, to block binding, thusmodulating (agonizing or antagonizing) the peptides activity. Antibodiescan be prepared against specific fragments containing sites required forfunction or against intact protein that is associated with a cell orcell membrane. See FIG. 2 for structural information relating to theproteins of the present invention.

[0095] The invention also encompasses kits for using antibodies todetect the presence of a protein in a biological sample. The kit cancomprise antibodies such as a labeled or labelable antibody and acompound or agent for detecting protein in a biological sample; meansfor determining the amount of protein in the sample; means for comparingthe amount of protein in the sample with a standard; and instructionsfor use. Such a kit can be supplied to detect a single protein orepitope or can be configured to detect one of a multitude of epitopes,such as in an antibody detection array. Arrays are described in detailbelow for nuleic acid arrays and similar methods have been developed forantibody arrays.

Nucleic Acid Molecules

[0096] The present invention further provides isolated nucleic acidmolecules that encode a kinase peptide or protein of the presentinvention (cDNA, transcript and genomic sequence). Such nucleic acidmolecules will consist of, consist essentially of, or comprise anucleotide sequence that encodes one of the kinase peptides of thepresent invention, an allelic variant thereof, or an ortholog or paralogthereof.

[0097] As used herein, an “isolated” nucleic acid molecule is one thatis separated from other nucleic acid present in the natural source ofthe nucleic acid. Preferably, an “isolated” nucleic acid is free ofsequences which naturally flank the nucleic acid (i.e., sequenceslocated at the 5′ and 3′ ends of the nucleic acid) in the genomic DNA ofthe organism from which the nucleic acid is derived. However, there canbe some flanking nucleotide sequences, for example up to about 5KB, 4KB,3KB, 2KB, or 1KB or less, particularly contiguous peptide encodingsequences and peptide encoding sequences within the same gene butseparated by introns in the genomic sequence. The important point isthat the nucleic acid is isolated from remote and unimportant flankingsequences such that it can be subjected to the specific manipulationsdescribed herein such as recombinant expression, preparation of probesand primers, and other uses specific to the nucleic acid sequences.

[0098] Moreover, an “isolated” nucleic acid molecule, such as atranscript/cDNA molecule, can be substantially free of other cellularmaterial, or culture medium when produced by recombinant techniques, orchemical precursors or other chemicals when chemically synthesized.However, the nucleic acid molecule can be fused to other coding orregulatory sequences and still be considered isolated.

[0099] For example, recombinant DNA molecules contained in a vector areconsidered isolated. Further examples of isolated DNA molecules includerecombinant DNA molecules maintained in heterologous host cells orpurified (partially or substantially) DNA molecules in solution.Isolated RNA molecules include in vivo or in vitro RNA transcripts ofthe isolated DNA molecules of the present invention. Isolated nucleicacid molecules according to the present invention further include suchmolecules produced synthetically.

[0100] Accordingly, the present invention provides nucleic acidmolecules that consist of the nucleotide sequence shown in FIG. 1 or 3(SEQ ID NO: 1, transcript sequence and SEQ ID NO: 3, genomic sequence),or any nucleic acid molecule that encodes the protein provided in FIG.2, SEQ ID NO: 2. A nucleic acid molecule consists of a nucleotidesequence when the nucleotide sequence is the complete nucleotidesequence of the nucleic acid molecule.

[0101] The present invention further provides nucleic acid moleculesthat consist essentially of the nucleotide sequence shown in FIG. 1 or 3(SEQ ID NO: 1, transcript sequence and SEQ ID NO: 3, genomic sequence),or any nucleic acid molecule that encodes the protein provided in FIG.2, SEQ ID NO: 2. A nucleic acid molecule consists essentially of anucleotide sequence when such a nucleotide sequence is present with onlya few additional nucleic acid residues in the final nucleic acidmolecule.

[0102] The present invention further provides nucleic acid moleculesthat comprise the nucleotide sequences shown in FIG. 1 or 3 (SEQ ID NO:1, transcript sequence and SEQ ID NO: 3, genomic sequence), or anynucleic acid molecule that encodes the protein provided in FIG. 2, SEQID NO: 2. A nucleic acid molecule comprises a nucleotide sequence whenthe nucleotide sequence is at least part of the final nucleotidesequence of the nucleic acid molecule. In such a fashion, the nucleicacid molecule can be only the nucleotide sequence or have additionalnucleic acid residues, such as nucleic acid residues that are naturallyassociated with it or heterologous nucleotide sequences. Such a nucleicacid molecule can have a few additional nucleotides or can comprisesseveral hundred or more additional nucleotides. A brief description ofhow various types of these nucleic acid molecules can be readilymade/isolated is provided below.

[0103] In FIGS. 1 and 3, both coding and non-coding sequences areprovided. Because of the source of the present invention, humans genomicsequence (FIG. 3) and cDNA/transcript sequences (FIG. 1), the nucleicacid molecules in the Figures will contain genomic intronic sequences,5′ and 3′ non-coding sequences, gene regulatory regions and non-codingintergenic sequences. In general such sequence features are either notedin FIGS. 1 and 3 or can readily be identified using computational toolsknown in the art. As discussed below, some of the non-coding regions,particularly gene regulatory elements such as promoters, are useful fora variety of purposes, e.g. control of heterologous gene expression,target for identifying gene activity modulating compounds, and areparticularly claimed as fragments of the genomic sequence providedherein.

[0104] The isolated nucleic acid molecules can encode the mature proteinplus additional amino or carboxyl-terminal amino acids, or amino acidsinterior to the mature peptide (when the mature form has more than onepeptide chain, for instance). Such sequences may play a role inprocessing of a protein from precursor to a mature form, facilitateprotein trafficking, prolong or shorten protein half-life or facilitatemanipulation of a protein for assay or production, among other things.As generally is the case in situ, the additional amino acids may beprocessed away from the mature protein by cellular enzymes.

[0105] As mentioned above, the isolated nucleic acid molecules include,but are not limited to, the sequence encoding the kinase peptide alone,the sequence encoding the mature peptide and additional codingsequences, such as a leader or secretory sequence (e.g., a pre-pro orpro-protein sequence), the sequence encoding the mature peptide, with orwithout the additional coding sequences, plus additional non-codingsequences, for example introns and non-coding 5′ and 3′ sequences suchas transcribed but non-translated sequences that play a role intranscription, mRNA processing (including splicing and polyadenylationsignals), ribosome binding and stability of mRNA. In addition, thenucleic acid molecule may be fused to a marker sequence encoding, forexample, a peptide that facilitates purification.

[0106] Isolated nucleic acid molecules can be in the form of RNA, suchas mRNA, or in the form DNA, including cDNA and genomic DNA obtained bycloning or produced by chemical synthetic techniques or by a combinationthereof. The nucleic acid, especially DNA, can be double-stranded orsingle-stranded. Single-stranded nucleic acid can be the coding strand(sense strand) or the non-coding strand (anti-sense strand).

[0107] The invention further provides nucleic acid molecules that encodefragments of the peptides of the present invention as well as nucleicacid molecules that encode obvious variants of the kinase proteins ofthe present invention that are described above. Such nucleic acidmolecules may be naturally occurring, such as allelic variants (samelocus), paralogs (different locus), and orthologs (different organism),or may be constructed by recombinant DNA methods or by chemicalsynthesis. Such non-naturally occurring variants may be made bymutagenesis techniques, including those applied to nucleic acidmolecules, cells, or organisms. Accordingly, as discussed above, thevariants can contain nucleotide substitutions, deletions, inversions andinsertions. Variation can occur in either or both the coding andnon-coding regions. The variations can produce both conservative andnon-conservative amino acid substitutions.

[0108] The present invention further provides non-coding fragments ofthe nucleic acid molecules provided in FIGS. 1 and 3. Preferrednon-coding fragments include, but are not limited to, promotersequences, enhancer sequences, gene modulating sequences and genetermination sequences. Such fragments are useful in controllingheterologous gene expression and in developing screens to identifygene-modulating agents. A promoter can readily be identified as being 5′to the ATG start site in the genomic sequence provided in FIG. 3.

[0109] A fragment comprises a contiguous nucleotide sequence greaterthan 12 or more nucleotides. Further, a fragment could at least 30, 40,50, 100, 250 or 500 nucleotides in length. The length of the fragmentwill be based on its intended use. For example, the fragment can encodeepitope bearing regions of the peptide, or can be useful as DNA probesand primers. Such fragments can be isolated using the known nucleotidesequence to synthesize an oligonucleotide probe. A labeled probe canthen be used to screen a cDNA library, genomic DNA library, or mRNA toisolate nucleic acid corresponding to the coding region. Further,primers can be used in PCR reactions to clone specific regions of gene.

[0110] A probe/primer typically comprises substantially a purifiedoligonucleotide or oligonucleotide pair. The oligonucleotide typicallycomprises a region of nucleotide sequence that hybridizes understringent conditions to at least about 12, 20, 25, 40, 50 or moreconsecutive nucleotides.

[0111] Orthologs, homologs, and allelic variants can be identified usingmethods well known in the art. As described in the Peptide Section,these variants comprise a nucleotide sequence encoding a peptide that istypically 60-70%, 70-80%, 80-90%, and more typically at least about90-95% or more homologous to the nucleotide sequence shown in the Figuresheets or a fragment of this sequence. Such nucleic acid molecules canreadily be identified as being able to hybridize under moderate tostringent conditions, to the nucleotide sequence shown in the Figuresheets or a fragment of the sequence. Allelic variants can readily bedetermined by genetic locus of the encoding gene. As indicated by thedata presented in FIG. 3, the map position was determined to be onchromosome 10 by ePCR.

[0112]FIG. 3 provides information on SNPs that have been found in thegene encoding the kinase protein of the present invention. SNPs wereidentified at 17 different nucleotide positions. SNPs such as these,particularly SNPs outside the ORF and in the first intron, may affectcontrol/regulatory elements.

[0113] As used herein, the term “hybridizes under stringent conditions”is intended to describe conditions for hybridization and washing underwhich nucleotide sequences encoding a peptide at least 60-70% homologousto each other typically remain hybridized to each other. The conditionscan be such that sequences at least about 60%, at least about 70%, or atleast about 80% or more homologous to each other typically remainhybridized to each other. Such stringent conditions are known to thoseskilled in the art and can be found in Current Protocols in MolecularBiology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6. One example ofstringent hybridization conditions are hybridization in 6× sodiumchloride/sodium citrate (SSC) at about 45C, followed by one or morewashes in 0.2×SSC, 0.1% SDS at 50-65C. Examples of moderate to lowstringency hybridization conditions are well known in the art.

Nucleic Acid Molecule Uses

[0114] The nucleic acid molecules of the present invention are usefulfor probes, primers, chemical intermediates, and in biological assays.The nucleic acid molecules are useful as a hybridization probe formessenger RNA, transcript/cDNA and genomic DNA to isolate full-lengthcDNA and genomic clones encoding the peptide described in FIG. 2 and toisolate cDNA and genomic clones that correspond to variants (alleles,orthologs, etc.) producing the same or related peptides shown in FIG. 2.As indicated by the data presented inAs illustrated in FIG. 3, SNPs wereidentified at 17 different nucleotide positions.

[0115] The probe can correspond to any sequence along the entire lengthof the nucleic acid molecules provided in the Figures. Accordingly, itcould be derived from 5′ noncoding regions, the coding region, and 3′noncoding regions. However, as discussed, fragments are not to beconstrued as encompassing fragments disclosed prior to the presentinvention.

[0116] The nucleic acid molecules are also useful as primers for PCR toamplify any given region of a nucleic acid molecule and are useful tosynthesize antisense molecules of desired length and sequence.

[0117] The nucleic acid molecules are also useful for constructingrecombinant vectors. Such vectors include expression vectors thatexpress a portion of, or all of, the peptide sequences. Vectors alsoinclude insertion vectors, used to integrate into another nucleic acidmolecule sequence, such as into the cellular genome, to alter in situexpression of a gene and/or gene product. For example, an endogenouscoding sequence can be replaced via homologous recombination with all orpart of the coding region containing one or more specifically introducedmutations.

[0118] The nucleic acid molecules are also useful for expressingantigenic portions of the proteins.

[0119] The nucleic acid molecules are also useful as probes fordetermining the chromosomal positions of the nucleic acid molecules bymeans of in situ hybridization methods. As indicated by the datapresented in FIG. 3, the map position was determined to be on chromosome10 by ePCR.

[0120] The nucleic acid molecules are also useful in making vectorscontaining the gene regulatory regions of the nucleic acid molecules ofthe present invention.

[0121] The nucleic acid molecules are also useful for designingribozymes corresponding to all, or a part, of the mRNA produced from thenucleic acid molecules described herein.

[0122] The nucleic acid molecules are also useful for making vectorsthat express part, or all, of the peptides.

[0123] The nucleic acid molecules are also useful for constructing hostcells expressing a part, or all, of the nucleic acid molecules andpeptides.

[0124] The nucleic acid molecules are also useful for constructingtransgenic animals expressing all, or a part, of the nucleic acidmolecules and peptides.

[0125] The nucleic acid molecules are also useful as hybridizationprobes for determining the presence, level, form and distribution ofnucleic acid expression. Experimental data as provided in FIG. 1indicates that kinase proteins of the present invention are expressed inhumans in prostate, lung, and whole brain, as indicated by virtualnorthern blot analysis. In addition, PCR-based tissue screening panelindicates expression in whole brain. Accordingly, the probes can be usedto detect the presence of, or to determine levels of, a specific nucleicacid molecule in cells, tissues, and in organisms. The nucleic acidwhose level is determined can be DNA or RNA. Accordingly, probescorresponding to the peptides described herein can be used to assessexpression and/or gene copy number in a given cell, tissue, or organism.These uses are relevant for diagnosis of disorders involving an increaseor decrease in kinase protein expression relative to normal results.

[0126] In vitro techniques for detection of mRNA include Northernhybridizations and in situ hybridizations. In vitro techniques fordetecting DNA includes Southern hybridizations and in situhybridization.

[0127] Probes can be used as a part of a diagnostic test kit foridentifying cells or tissues that express a kinase protein, such as bymeasuring a level of a kinase-encoding nucleic acid in a sample of cellsfrom a subject e.g., mRNA or genomic DNA, or determining if a kinasegene has been mutated. Experimental data as provided in FIG. 1 indicatesthat kinase proteins of the present invention are expressed in humans inprostate, lung, and whole brain, as indicated by virtual northern blotanalysis. In addition, PCR-based tissue screening panel indicatesexpression in whole brain.

[0128] Nucleic acid expression assays are useful for drug screening toidentify compounds that modulate kinase nucleic acid expression.

[0129] The invention thus provides a method for identifying a compoundthat can be used to treat a disorder associated with nucleic acidexpression of the kinase gene, particularly biological and pathologicalprocesses that are mediated by the kinase in cells and tissues thatexpress it. Experimental data as provided in FIG. 1 indicates expressionin humans in prostate, lung, and whole brain. The method typicallyincludes assaying the ability of the compound to modulate the expressionof the kinase nucleic acid and thus identifying a compound that can beused to treat a disorder characterized by undesired kinase nucleic acidexpression. The assays can be performed in cell-based and cell-freesystems. Cell-based assays include cells naturally expressing the kinasenucleic acid or recombinant cells genetically engineered to expressspecific nucleic acid sequences.

[0130] The assay for kinase nucleic acid expression can involve directassay of nucleic acid levels, such as mRNA levels, or on collateralcompounds involved in the signal pathway. Further, the expression ofgenes that are up- or down-regulated in response to the kinase proteinsignal pathway can also be assayed. In this embodiment the regulatoryregions of these genes can be operably linked to a reporter gene such asluciferase.

[0131] Thus, modulators of kinase gene expression can be identified in amethod wherein a cell is contacted with a candidate compound and theexpression of mRNA determined. The level of expression of kinase mRNA inthe presence of the candidate compound is compared to the level ofexpression of kinase mRNA in the absence of the candidate compound. Thecandidate compound can then be identified as a modulator of nucleic acidexpression based on this comparison and be used, for example to treat adisorder characterized by aberrant nucleic acid expression. Whenexpression of mRNA is statistically significantly greater in thepresence of the candidate compound than in its absence, the candidatecompound is identified as a stimulator of nucleic acid expression. Whennucleic acid expression is statistically significantly less in thepresence of the candidate compound than in its absence, the candidatecompound is identified as an inhibitor of nucleic acid expression.

[0132] The invention further provides methods of treatment, with thenucleic acid as a target, using a compound identified through drugscreening as a gene modulator to modulate kinase nucleic acid expressionin cells and tissues that express the kinase. Experimental data asprovided in FIG. 1 indicates that kinase proteins of the presentinvention are expressed in humans in prostate, lung, and whole brain, asindicated by virtual northern blot analysis. In addition, PCR-basedtissue screening panel indicates expression in whole brain. Modulationincludes both up-regulation (i.e. activation or agonization) ordown-regulation (suppression or antagonization) or nucleic acidexpression.

[0133] Alternatively, a modulator for kinase nucleic acid expression canbe a small molecule or drug identified using the screening assaysdescribed herein as long as the drug or small molecule inhibits thekinase nucleic acid expression in the cells and tissues that express theprotein. Experimental data as provided in FIG. 1 indicates expression inhumans in prostate, lung, and whole brain.

[0134] The nucleic acid molecules are also useful for monitoring theeffectiveness of modulating compounds on the expression or activity ofthe kinase gene in clinical trials or in a treatment regimen. Thus, thegene expression pattern can serve as a barometer for the continuingeffectiveness of treatment with the compound, particularly withcompounds to which a patient can develop resistance. The gene expressionpattern can also serve as a marker indicative of a physiologicalresponse of the affected cells to the compound. Accordingly, suchmonitoring would allow either increased administration of the compoundor the administration of alternative compounds to which the patient hasnot become resistant. Similarly, if the level of nucleic acid expressionfalls below a desirable level, administration of the compound could becommensurately decreased.

[0135] The nucleic acid molecules are also useful in diagnostic assaysfor qualitative changes in kinase nucleic acid expression, andparticularly in qualitative changes that lead to pathology. The nucleicacid molecules can be used to detect mutations in kinase genes and geneexpression products such as mRNA. The nucleic acid molecules can be usedas hybridization probes to detect naturally occurring genetic mutationsin the kinase gene and thereby to determine whether a subject with themutation is at risk for a disorder caused by the mutation. Mutationsinclude deletion, addition, or substitution of one or more nucleotidesin the gene, chromosomal rearrangement, such as inversion ortransposition, modification of genomic DNA, such as aberrant methylationpatterns or changes in gene copy number, such as amplification.Detection of a mutated form of the kinase gene associated with adysfunction provides a diagnostic tool for an active disease orsusceptibility to disease when the disease results from overexpression,underexpression, or altered expression of a kinase protein.

[0136] Individuals carrying mutations in the kinase gene can be detectedat the nucleic acid level by a variety of techniques. FIG. 3 providesinformation on SNPs that have been found in the gene encoding the kinaseprotein of the present invention. SNPs were identified at 17 differentnucleotide positions. SNPs such as these, particularly SNPs outside theORF and in the first intron, may affect control/regulatory elements. Asindicated by the data presented in FIG. 3, the map position wasdetermined to be on chromosome 10 by ePCR. Genomic DNA can be analyzeddirectly or can be amplified by using PCR prior to analysis. RNA or cDNAcan be used in the same way. In some uses, detection of the mutationinvolves the use of a probe/primer in a polymerase chain reaction (PCR)(see, e.g. U.S. Pat. Nos. 4,683,195 and 4,683,202), such as anchor PCRor RACE PCR, or, alternatively, in a ligation chain reaction (LCR) (see,e.g., Landegran et al., Science 241:1077-1080 (1988); and Nakazawa etal., PNAS 91:360-364 (1994)), the latter of which can be particularlyuseful for detecting point mutations in the gene (see Abravaya et al.,Nucleic Acids Res. 23:675-682 (1995)). This method can include the stepsof collecting a sample of cells from a patient, isolating nucleic acid(e.g., genomic, mRNA or both) from the cells of the sample, contactingthe nucleic acid sample with one or more primers which specificallyhybridize to a gene under conditions such that hybridization andamplification of the gene (if present) occurs, and detecting thepresence or absence of an amplification product, or detecting the sizeof the amplification product and comparing the length to a controlsample. Deletions and insertions can be detected by a change in size ofthe amplified product compared to the normal genotype. Point mutationscan be identified by hybridizing amplified DNA to normal RNA orantisense DNA sequences.

[0137] Alternatively, mutations in a kinase gene can be directlyidentified, for example, by alterations in restriction enzyme digestionpatterns determined by gel electrophoresis.

[0138] Further, sequence-specific ribozymes (U.S. Pat. No. 5,498,531)can be used to score for the presence of specific mutations bydevelopment or loss of a ribozyme cleavage site. Perfectly matchedsequences can be distinguished from mismatched sequences by nucleasecleavage digestion assays or by differences in melting temperature.

[0139] Sequence changes at specific locations can also be assessed bynuclease protection assays such as RNase and S1 protection or thechemical cleavage method. Furthermore, sequence differences between amutant kinase gene and a wild-type gene can be determined by direct DNAsequencing. A variety of automated sequencing procedures can be utilizedwhen performing the diagnostic assays (Naeve, C. W., (1995)Biotechniques 19:448), including sequencing by mass spectrometry (see,e.g., PCT International Publication No. WO 94/16101; Cohen et al., Adv.Chromatogr. 36:127-162 (1996); and Griffin et al., Appl. Biochem.Biotechnol. 38:147-159 (1993)).

[0140] Other methods for detecting mutations in the gene include methodsin which protection from cleavage agents is used to detect mismatchedbases in RNA/RNA or RNA/DNA duplexes (Myers et al., Science 230:1242(1985)); Cotton et al., PNAS 85:4397 (1988); Saleeba et al., Meth.Enzymol. 217:286-295 (1992)), electrophoretic mobility of mutant andwild type nucleic acid is compared (Orita et al., PNAS 86:2766 (1989);Cotton et al., Mutat. Res. 285:125-144 (1993); and Hayashi et al.,Genet. Anal. Tech. Appl. 9:73-79 (1992)), and movement of mutant orwild-type fragments in polyacrylamide gels containing a gradient ofdenaturant is assayed using denaturing gradient gel electrophoresis(Myers et al., Nature 313:495 (1985)). Examples of other techniques fordetecting point mutations include selective oligonucleotidehybridization, selective amplification, and selective primer extension.

[0141] The nucleic acid molecules are also useful for testing anindividual for a genotype that while not necessarily causing thedisease, nevertheless affects the treatment modality. Thus, the nucleicacid molecules can be used to study the relationship between anindividual's genotype and the individual's response to a compound usedfor treatment (pharmacogenomic relationship). Accordingly, the nucleicacid molecules described herein can be used to assess the mutationcontent of the kinase gene in an individual in order to select anappropriate compound or dosage regimen for treatment. FIG. 3 providesinformation on SNPs that have been found in the gene encoding the kinaseprotein of the present invention. SNPs were identified at 17 differentnucleotide positions. SNPs such as these, particularly SNPs outside theORF and in the first intron, may affect control/regulatory elements.

[0142] Thus nucleic acid molecules displaying genetic variations thataffect treatment provide a diagnostic target that can be used to tailortreatment in an individual. Accordingly, the production of recombinantcells and animals containing these polymorphisms allow effectiveclinical design of treatment compounds and dosage regimens.

[0143] The nucleic acid molecules are thus useful as antisenseconstructs to control kinase gene expression in cells, tissues, andorganisms. A DNA antisense nucleic acid molecule is designed to becomplementary to a region of the gene involved in transcription,preventing transcription and hence production of kinase protein. Anantisense RNA or DNA nucleic acid molecule would hybridize to the mRNAand thus block translation of mRNA into kinase protein.

[0144] Alternatively, a class of antisense molecules can be used toinactivate mRNA in order to decrease expression of kinase nucleic acid.Accordingly, these molecules can treat a disorder characterized byabnormal or undesired kinase nucleic acid expression. This techniqueinvolves cleavage by means of ribozymes containing nucleotide sequencescomplementary to one or more regions in the mRNA that attenuate theability of the mRNA to be translated. Possible regions include codingregions and particularly coding regions corresponding to the catalyticand other functional activities of the kinase protein, such as substratebinding.

[0145] The nucleic acid molecules also provide vectors for gene therapyin patients containing cells that are aberrant in kinase geneexpression. Thus, recombinant cells, which include the patient's cellsthat have been engineered ex vivo and returned to the patient, areintroduced into an individual where the cells produce the desired kinaseprotein to treat the individual.

[0146] The invention also encompasses kits for detecting the presence ofa kinase nucleic acid in a biological sample. Experimental data asprovided in FIG. 1 indicates that kinase proteins of the presentinvention are expressed in humans in prostate, lung, and whole brain, asindicated by virtual northern blot analysis. In addition, PCR-basedtissue screening panel indicates expression in whole brain. For example,the kit can comprise reagents such as a labeled or labelable nucleicacid or agent capable of detecting kinase nucleic acid in a biologicalsample; means for determining the amount of kinase nucleic acid in thesample; and means for comparing the amount of kinase nucleic acid in thesample with a standard. The compound or agent can be packaged in asuitable container. The kit can further comprise instructions for usingthe kit to detect kinase protein mRNA or DNA.

Nucleic Acid Arrays

[0147] The present invention further provides nucleic acid detectionkits, such as arrays or microarrays of nucleic acid molecules that arebased on the sequence information provided in FIGS. 1 and 3 (SEQ IDNOS:1 and 3).

[0148] As used herein “Arrays” or “Microarrays” refers to an array ofdistinct polynucleotides or oligonucleotides synthesized on a substrate,such as paper, nylon or other type of membrane, filter, chip, glassslide, or any other suitable solid support. In one embodiment, themicroarray is prepared and used according to the methods described inU.S. Pat. 5,837,832, Chee et al., PCT application W095/11995 (Chee etal.), Lockhart, D. J. et al. (1996; Nat. Biotech. 14: 1675-1680) andSchena, M. et al. (1996; Proc. Natl. Acad. Sci. 93: 10614-10619), all ofwhich are incorporated herein in their entirety by reference. In otherembodiments, such arrays are produced by the methods described by Brownet al., U.S. Pat. No. 5,807,522.

[0149] The microarray or detection kit is preferably composed of a largenumber of unique, single-stranded nucleic acid sequences, usually eithersynthetic antisense oligonucleotides or fragments of cDNAs, fixed to asolid support. The oligonucleotides are preferably about 6-60nucleotides in length, more preferably 15-30 nucleotides in length, andmost preferably about 20-25 nucleotides in length. For a certain type ofmicroarray or detection kit, it may be preferable to useoligonucleotides that are only 7-20 nucleotides in length. Themicroarray or detection kit may contain oligonucleotides that cover theknown 5′, or 3′, sequence, sequential oligonucleotides which cover thefull length sequence; or unique oligonucleotides selected fromparticular areas along the length of the sequence. Polynucleotides usedin the microarray or detection kit may be oligonucleotides that arespecific to a gene or genes of interest.

[0150] In order to produce oligonucleotides to a known sequence for amicroarray or detection kit, the gene(s) of interest (or an ORFidentified from the contigs of the present invention) is typicallyexamined using a computer algorithm which starts at the 5′ or at the 3′end of the nucleotide sequence. Typical algorithms will then identifyoligomers of defined length that are unique to the gene, have a GCcontent within a range suitable for hybridization, and lack predictedsecondary structure that may interfere with hybridization. In certainsituations it may be appropriate to use pairs of oligonucleotides on amicroarray or detection kit. The “pairs” will be identical, except forone nucleotide that preferably is located in the center of the sequence.The second oligonucleotide in the pair (mismatched by one) serves as acontrol. The number of oligonucleotide pairs may range from two to onemillion. The oligomers are synthesized at designated areas on asubstrate using a light-directed chemical process. The substrate may bepaper, nylon or other type of membrane, filter, chip, glass slide or anyother suitable solid support.

[0151] In another aspect, an oligonucleotide may be synthesized on thesurface of the substrate by using a chemical coupling procedure and anink jet application apparatus, as described in PCT application W095/251116 (Baldeschweiler et al.) which is incorporated herein in itsentirety by reference. In another aspect, a “gridded” array analogous toa dot (or slot) blot may be used to arrange and link cDNA fragments oroligonucleotides to the surface of a substrate using a vacuum system,thermal, UV, mechanical or chemical bonding procedures. An array, suchas those described above, may be produced by hand or by using availabledevices (slot blot or dot blot apparatus), materials (any suitable solidsupport), and machines (including robotic instruments), and may contain8, 24, 96, 384, 1536, 6144 or more oligonucleotides, or any other numberbetween two and one million which lends itself to the efficient use ofcommercially available instrumentation.

[0152] In order to conduct sample analysis using a microarray ordetection kit, the RNA or DNA from a biological sample is made intohybridization probes. The mRNA is isolated, and cDNA is produced andused as a template to make antisense RNA (aRNA). The aRNA is amplifiedin the presence of fluorescent nucleotides, and labeled probes areincubated with the microarray or detection kit so that the probesequences hybridize to complementary oligonucleotides of the microarrayor detection kit. Incubation conditions are adjusted so thathybridization occurs with precise complementary matches or with variousdegrees of less complementarity. After removal of nonhybridized probes,a scanner is used to determine the levels and patterns of fluorescence.The scanned images are examined to determine degree of complementarityand the relative abundance of each oligonucleotide sequence on themicroarray or detection kit. The biological samples may be obtained fromany bodily fluids (such as blood, urine, saliva, phlegm, gastric juices,etc.), cultured cells, biopsies, or other tissue preparations. Adetection system may be used to measure the absence, presence, andamount of hybridization for all of the distinct sequencessimultaneously. This data may be used for large-scale correlationstudies on the sequences, expression patterns, mutations, variants, orpolymorphisms among samples.

[0153] Using such arrays, the present invention provides methods toidentify the expression of the kinase proteins/peptides of the presentinvention. In detail, such methods comprise incubating a test samplewith one or more nucleic acid molecules and assaying for binding of thenucleic acid molecule with components within the test sample. Suchassays will typically involve arrays comprising many genes, at least oneof which is a gene of the present invention and or alleles of the kinasegene of the present invention. FIG. 3 provides information on SNPs thathave been found in the gene encoding the kinase protein of the presentinvention. SNPs were identified at 17 different nucleotide positions.SNPs such as these, particularly SNPs outside the ORF and in the firstintron, may affect control/regulatory elements.

[0154] Conditions for incubating a nucleic acid molecule with a testsample vary. Incubation conditions depend on the format employed in theassay, the detection methods employed, and the type and nature of thenucleic acid molecule used in the assay. One skilled in the art willrecognize that any one of the commonly available hybridization,amplification or array assay formats can readily be adapted to employthe novel fragments of the Human genome disclosed herein. Examples ofsuch assays can be found in Chard, T, An Introduction toRadioimmunoassay and Related Techniques, Elsevier Science Publishers,Amsterdam, The Netherlands (1986); Bullock, G. R. et al., Techniques inImmunocytochemistry, Academic Press, Orlando, Fla. Vol. 1 (1982), Vol. 2(1983), Vol. 3 (1985); Tijssen, P., Practice and Theory of EnzymeImmunoassays: Laboratory Techniques in Biochemistry and MolecularBiology, Elsevier Science Publishers, Amsterdam, The Netherlands (1985).

[0155] The test samples of the present invention include cells, proteinor membrane extracts of cells. The test sample used in theabove-described method will vary based on the assay format, nature ofthe detection method and the tissues, cells or extracts used as thesample to be assayed. Methods for preparing nucleic acid extracts or ofcells are well known in the art and can be readily be adapted in orderto obtain a sample that is compatible with the system utilized.

[0156] In another embodiment of the present invention, kits are providedwhich contain the necessary reagents to carry out the assays of thepresent invention.

[0157] Specifically, the invention provides a compartmentalized kit toreceive, in close confinement, one or more containers which comprises:(a) a first container comprising one of the nucleic acid molecules thatcan bind to a fragment of the Human genome disclosed herein; and (b) oneor more other containers comprising one or more of the following: washreagents, reagents capable of detecting presence of a bound nucleicacid.

[0158] In detail, a compartmentalized kit includes any kit in whichreagents are contained in separate containers. Such containers includesmall glass containers, plastic containers, strips of plastic, glass orpaper, or arraying material such as silica. Such containers allows oneto efficiently transfer reagents from one compartment to anothercompartment such that the samples and reagents are notcross-contaminated, and the agents or solutions of each container can beadded in a quantitative fashion from one compartment to another. Suchcontainers will include a container which will accept the test sample, acontainer which contains the nucleic acid probe, containers whichcontain wash reagents (such as phosphate buffered saline, Tris-buffers,etc.), and containers which contain the reagents used to detect thebound probe. One skilled in the art will readily recognize that thepreviously unidentified kinase gene of the present invention can beroutinely identified using the sequence information disclosed herein canbe readily incorporated into one of the established kit formats whichare well known in the art, particularly expression arrays.

Vectors/Host Cells

[0159] The invention also provides vectors containing the nucleic acidmolecules described herein. The term “vector” refers to a vehicle,preferably a nucleic acid molecule, which can transport the nucleic acidmolecules. When the vector is a nucleic acid molecule, the nucleic acidmolecules are covalently linked to the vector nucleic acid. With thisaspect of the invention, the vector includes a plasmid, single or doublestranded phage, a single or double stranded RNA or DNA viral vector, orartificial chromosome, such as a BAC, PAC, YAC, OR MAC.

[0160] A vector can be maintained in the host cell as anextrachromosomal element where it replicates and produces additionalcopies of the nucleic acid molecules. Alternatively, the vector mayintegrate into the host cell genome and produce additional copies of thenucleic acid molecules when the host cell replicates.

[0161] The invention provides vectors for the maintenance (cloningvectors) or vectors for expression (expression vectors) of the nucleicacid molecules. The vectors can function in prokaryotic or eukaryoticcells or in both (shuttle vectors).

[0162] Expression vectors contain cis-acting regulatory regions that areoperably linked in the vector to the nucleic acid molecules such thattranscription of the nucleic acid molecules is allowed in a host cell.The nucleic acid molecules can be introduced into the host cell with aseparate nucleic acid molecule capable of affecting transcription. Thus,the second nucleic acid molecule may provide a trans-acting factorinteracting with the cis-regulatory control region to allowtranscription of the nucleic acid molecules from the vector.Alternatively, a trans-acting factor may be supplied by the host cell.Finally, a trans-acting factor can be produced from the vector itself.It is understood, however, that in some embodiments, transcriptionand/or translation of the nucleic acid molecules can occur in acell-free system.

[0163] The regulatory sequence to which the nucleic acid moleculesdescribed herein can be operably linked include promoters for directingmRNA transcription. These include, but are not limited to, the leftpromoter from bacteriophage λ, the lac, TRP, and TAC promoters from E.coli, the early and late promoters from SV40, the CMV immediate earlypromoter, the adenovirus early and late promoters, and retroviruslong-terminal repeats.

[0164] In addition to control regions that promote transcription,expression vectors may also include regions that modulate transcription,such as repressor binding sites and enhancers. Examples include the SV40enhancer, the cytomegalovirus immediate early enhancer, polyomaenhancer, adenovirus enhancers, and retrovirus LTR enhancers.

[0165] In addition to containing sites for transcription initiation andcontrol, expression vectors can also contain sequences necessary fortranscription termination and, in the transcribed region a ribosomebinding site for translation. Other regulatory control elements forexpression include initiation and termination codons as well aspolyadenylation signals. The person of ordinary skill in the art wouldbe aware of the numerous regulatory sequences that are useful inexpression vectors. Such regulatory sequences are described, forexample, in Sambrook et al., Molecular Cloning: A Laboratory Manual.2nd. ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,(1989).

[0166] A variety of expression vectors can be used to express a nucleicacid molecule. Such vectors include chromosomal, episomal, andvirus-derived vectors, for example vectors derived from bacterialplasmids, from bacteriophage, from yeast episomes, from yeastchromosomal elements, including yeast artificial chromosomes, fromviruses such as baculoviruses, papovaviruses such as SV40, Vacciniaviruses, adenoviruses, poxviruses, pseudorabies viruses, andretroviruses. Vectors may also be derived from combinations of thesesources such as those derived from plasrnid and bacteriophage geneticelements, e.g. cosmids and phagemids. Appropriate cloning and expressionvectors for prokaryotic and eukaryotic hosts are described in Sambrooket al., Molecular Cloning: A Laboratory Manual. 2nd. ed., Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., (1989).

[0167] The regulatory sequence may provide constitutive expression inone or more host cells (i.e. tissue specific) or may provide forinducible expression in one or more cell types such as by temperature,nutrient additive, or exogenous factor such as a hormone or otherligand. A variety of vectors providing for constitutive and inducibleexpression in prokaryotic and eukaryotic hosts are well known to thoseof ordinary skill in the art.

[0168] The nucleic acid molecules can be inserted into the vectornucleic acid by well-known methodology. Generally, the DNA sequence thatwill ultimately be expressed is joined to an expression vector bycleaving the DNA sequence and the expression vector with one or morerestriction enzymes and then ligating the fragments together. Proceduresfor restriction enzyme digestion and ligation are well known to those ofordinary skill in the art.

[0169] The vector containing the appropriate nucleic acid molecule canbe introduced into an appropriate host cell for propagation orexpression using well-known techniques. Bacterial cells include, but arenot limited to, E. coli, Streptomyces, and Salmonella typhimurium.Eukaryotic cells include, but are not limited to, yeast, insect cellssuch as Drosophila, animal cells such as COS and CHO cells, and plantcells.

[0170] As described herein, it may be desirable to express the peptideas a fusion protein. Accordingly, the invention provides fusion vectorsthat allow for the production of the peptides. Fusion vectors canincrease the expression of a recombinant protein, increase thesolubility of the recombinant protein, and aid in the purification ofthe protein by acting for example as a ligand for affinity purification.A proteolytic cleavage site may be introduced at the junction of thefusion moiety so that the desired peptide can ultimately be separatedfrom the fusion moiety. Proteolytic enzymes include, but are not limitedto, factor Xa, thrombin, and enterokinase. Typical fusion expressionvectors include pGEX (Smith et al., Gene 67:31-40 (1988)), pMAL (NewEngland Biolabs, Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.)which fuse glutathione S-transferase (GST), maltose E binding protein,or protein A, respectively, to the target recombinant protein. Examplesof suitable inducible non-fusion E. coli expression vectors include pTrc(Amann et al., Gene 69:301-315 (1988)) and pET 11d (Studier et al., GeneExpression Technology: Methods in Enzymology 185:60-89 (1990)).

[0171] Recombinant protein expression can be maximized in host bacteriaby providing a genetic background wherein the host cell has an impairedcapacity to proteolytically cleave the recombinant protein. (Gottesman,S., Gene Expression Technology: Methods in Enzymology 185, AcademicPress, San Diego, Calif. (1990)119-128). Alternatively, the sequence ofthe nucleic acid molecule of interest can be altered to providepreferential codon usage for a specific host cell, for example E. coli.(Wada et al., Nucleic Acids Res. 20:2111-2118 (1992)).

[0172] The nucleic acid molecules can also be expressed by expressionvectors that are operative in yeast. Examples of vectors for expressionin yeast e.g., S. cerevisiae include pYepSec1 (Baldari, et al., EMBO J.6:229-234 (1987)), pMFa (Kujan et al., Cell 30:933-943(1982)), pJRY88(Schultz et al., Gene 54:113-123 (1987)), and pYES2 (InvitrogenCorporation, San Diego, Calif.).

[0173] The nucleic acid molecules can also be expressed in insect cellsusing, for example, baculovirus expression vectors. Baculovirus vectorsavailable for expression of proteins in cultured insect cells (e.g., Sf9cells) include the pAc series (Smith et al., Mol. Cell Biol. 3:2156-2165(1983)) and the pVL series (Lucklow et al., Virology 170:31-39 (1989)).

[0174] In certain embodiments of the invention, the nucleic acidmolecules described herein are expressed in mammalian cells usingmammalian expression vectors. Examples of mammalian expression vectorsinclude pCDM8 (Seed, B. Nature 329:840(1987)) and pMT2PC (Kaufman etal., EMBO J. 6:187-195 (1987)).

[0175] The expression vectors listed herein are provided by way ofexample only of the well-known vectors available to those of ordinaryskill in the art that would be useful to express the nucleic acidmolecules. The person of ordinary skill in the art would be aware ofother vectors suitable for maintenance propagation or expression of thenucleic acid molecules described herein. These are found for example inSambrook, J., Fritsh, E. F., and Maniatis, T. Molecular Cloning: ALaboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., 1989.

[0176] The invention also encompasses vectors in which the nucleic acidsequences described herein are cloned into the vector in reverseorientation, but operably linked to a regulatory sequence that permitstranscription of antisense RNA. Thus, an antisense transcript can beproduced to all, or to a portion, of the nucleic acid molecule sequencesdescribed herein, including both coding and non-coding regions.Expression of this antisense RNA is subject to each of the parametersdescribed above in relation to expression of the sense RNA (regulatorysequences, constitutive or inducible expression, tissue-specificexpression).

[0177] The invention also relates to recombinant host cells containingthe vectors described herein. Host cells therefore include prokaryoticcells, lower eukaryotic cells such as yeast, other eukaryotic cells suchas insect cells, and higher eukaryotic cells such as mammalian cells.

[0178] The recombinant host cells are prepared by introducing the vectorconstructs described herein into the cells by techniques readilyavailable to the person of ordinary skill in the art. These include, butare not limited to, calcium phosphate transfection,DEAE-dextran-mediated transfection, cationic lipid-mediatedtransfection, electroporation, transduction, infection, lipofection, andother techniques such as those found in Sambrook, et al. (MolecularCloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989).

[0179] Host cells can contain more than one vector. Thus, differentnucleotide sequences can be introduced on different vectors of the samecell. Similarly, the nucleic acid molecules can be introduced eitheralone or with other nucleic acid molecules that are not related to thenucleic acid molecules such as those providing trans-acting factors forexpression vectors. When more than one vector is introduced into a cell,the vectors can be introduced independently, co-introduced or joined tothe nucleic acid molecule vector.

[0180] In the case of bacteriophage and viral vectors, these can beintroduced into cells as packaged or encapsulated virus by standardprocedures for infection and transduction. Viral vectors can bereplication-competent or replication-defective. In the case in whichviral replication is defective, replication will occur in host cellsproviding functions that complement the defects.

[0181] Vectors generally include selectable markers that enable theselection of the subpopulation of cells that contain the recombinantvector constructs. The marker can be contained in the same vector thatcontains the nucleic acid molecules described herein or may be on aseparate vector. Markers include tetracycline or ampicillin-resistancegenes for prokaryotic host cells and dihydrofolate reductase or neomycinresistance for eukaryotic host cells. However, any marker that providesselection for a phenotypic trait will be effective.

[0182] While the mature proteins can be produced in bacteria, yeast,mammalian cells, and other cells under the control of the appropriateregulatory sequences, cell-free transcription and translation systemscan also be used to produce these proteins using RNA derived from theDNA constructs described herein.

[0183] Where secretion of the peptide is desired, which is difficult toachieve with multi-transmembrane domain containing proteins such askinases, appropriate secretion signals are incorporated into the vector.The signal sequence can be endogenous to the peptides or heterologous tothese peptides.

[0184] Where the peptide is not secreted into the medium, which istypically the case with kinases, the protein can be isolated from thehost cell by standard disruption procedures, including freeze thaw,sonication, mechanical disruption, use of lysing agents and the like.The peptide can then be recovered and purified by well-knownpurification methods including ammonium sulfate precipitation, acidextraction, anion or cationic exchange chromatography, phosphocellulosechromatography, hydrophobic-interaction chromatography, affinitychromatography, hydroxylapatite chromatography, lectin chromatography,or high performance liquid chromatography.

[0185] It is also understood that depending upon the host cell inrecombinant production of the peptides described herein, the peptidescan have various glycosylation patterns, depending upon the cell, ormaybe non-glycosylated as when produced in bacteria. In addition, thepeptides may include an initial modified methionine in some cases as aresult of a host-mediated process.

Uses of Vectors and Host Cells

[0186] The recombinant host cells expressing the peptides describedherein have a variety of uses. First, the cells are useful for producinga kinase protein or peptide that can be further purified to producedesired amounts of kinase protein or fragments. Thus, host cellscontaining expression vectors are useful for peptide production.

[0187] Host cells are also useful for conducting cell-based assaysinvolving the kinase protein or kinase protein fragments, such as thosedescribed above as well as other formats known in the art. Thus, arecombinant host cell expressing a native kinase protein is useful forassaying compounds that stimulate or inhibit kinase protein function.

[0188] Host cells are also useful for identifying kinase protein mutantsin which these functions are affected. If the mutants naturally occurand give rise to a pathology, host cells containing the mutations areuseful to assay compounds that have a desired effect on the mutantkinase protein (for example, stimulating or inhibiting function) whichmay not be indicated by their effect on the native kinase protein.

[0189] Genetically engineered host cells can be further used to producenon-human transgenic animals. A transgenic animal is preferably amammal, for example a rodent, such as a rat or mouse, in which one ormore of the cells of the animal include a transgene. A transgene isexogenous DNA which is integrated into the genome of a cell from which atransgenic animal develops and which remains in the genome of the matureanimal in one or more cell types or tissues of the transgenic animal.These animals are useful for studying the function of a kinase proteinand identifying and evaluating modulators of kinase protein activity.Other examples of transgenic animals include non-human primates, sheep,dogs, cows, goats, chickens, and amphibians.

[0190] A transgenic animal can be produced by introducing nucleic acidinto the male pronuclei of a fertilized oocyte, e.g., by microinjection,retroviral infection, and allowing the oocyte to develop in apseudopregnant female foster animal. Any of the kinase proteinnucleotide sequences can be introduced as a transgene into the genome ofa non-human animal, such as a mouse.

[0191] Any of the regulatory or other sequences useful in expressionvectors can form part of the transgenic sequence. This includes intronicsequences and polyadenylation signals, if not already included. Atissue-specific regulatory sequence(s) can be operably linked to thetransgene to direct expression of the kinase protein to particularcells.

[0192] Methods for generating transgenic animals via embryo manipulationand microinjection, particularly animals such as mice, have becomeconventional in the art and are described, for example, in U.S. Pat.Nos. 4,736,866 and 4,870,009, both by Leder et al., U.S. Pat. No.4,873,191 by Wagner et al. and in Hogan, B., Manipulating the MouseEmbryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,1986). Similar methods are used for production of other transgenicanimals. A transgenic founder animal can be identified based upon thepresence of the transgene in its genome and/or expression of transgenicmRNA in tissues or cells of the animals. A transgenic founder animal canthen be used to breed additional animals carrying the transgene.Moreover, transgenic animals carrying a transgene can further be bred toother transgenic animals carrying other transgenes. A transgenic animalalso includes animals in which the entire animal or tissues in theanimal have been produced using the homologously recombinant host cellsdescribed herein.

[0193] In another embodiment, transgenic non-human animals can beproduced which contain selected systems that allow for regulatedexpression of the transgene. One example of such a system is thecre/loxP recombinase system of bacteriophage P1. For a description ofthe cre/loxP recombinase system, see, e.g., Lakso et al. PNAS89:6232-6236 (1992). Another example of a recombinase system is the FLPrecombinase system of S. cerevisiae (O'Gorman et al. Science251:1351-1355 (1991). If a cre/loxP recombinase system is used toregulate expression of the transgene, animals containing transgenesencoding both the Cre recombinase and a selected protein is required.Such animals can be provided through the construction of “double”transgenic animals, e.g., by mating two transgenic animals, onecontaining a transgene encoding a selected protein and the othercontaining a transgene encoding a recombinase.

[0194] Clones of the non-human transgenic animals described herein canalso be produced according to the methods described in Wilmut, I. et al.Nature 385:810-813 (1997) and PCT International Publication Nos. WO97/07668 and WO 97/07669. In brief, a cell, e.g., a somatic cell, fromthe transgenic animal can be isolated and induced to exit the growthcycle and enter G_(o) phase. The quiescent cell can then be fused, e.g.,through the use of electrical pulses, to an enucleated oocyte from ananimal of the same species from which the quiescent cell is isolated.The reconstructed oocyte is then cultured such that it develops tomorula or blastocyst and then transferred to pseudopregnant femalefoster animal. The offspring born of this female foster animal will be aclone of the animal from which the cell, e.g., the somatic cell, isisolated.

[0195] Transgenic animals containing recombinant cells that express thepeptides described herein are useful to conduct the assays describedherein in an in vivo context. Accordingly, the various physiologicalfactors that are present in vivo and that could effect substratebinding, kinase protein activation, and signal transduction, may not beevident from in vitro cell-free or cell-based assays. Accordingly, it isuseful to provide non-human transgenic animals to assay in vivo kinaseprotein function, including substrate interaction, the effect ofspecific mutant kinase proteins on kinase protein function and substrateinteraction, and the effect of chimeric kinase proteins. It is alsopossible to assess the effect of null mutations, that is, mutations thatsubstantially or completely eliminate one or more kinase proteinfunctions.

[0196] All publications and patents mentioned in the above specificationare herein incorporated by reference. Various modifications andvariations of the described method and system of the invention will beapparent to those skilled in the art without departing from the scopeand spirit of the invention. Although the invention has been describedin connection with specific preferred embodiments, it should beunderstood that the invention as claimed should not be unduly limited tosuch specific embodiments. Indeed, various modifications of theabove-described modes for carrying out the invention which are obviousto those skilled in the field of molecular biology or related fields areintended to be within the scope of the following claims.

1 5 1 1864 DNA Homo sapiens 1 cgagtgcgct gctgagtcct gtagataaagccgccaaccc cggggactgg tgtctcctga 60 gtgaccgtgc agccgtgggc gccatagaaagcagagaagg cagtgaactt cgaccacttc 120 cagatccttc gggccattgg gaagggcagctttggcaagg tgtgcattgt gcagaagcgg 180 gacacggaga agatgtacgc catgaagtacatgaacaagc agcagtgcat cgagcgcgac 240 gaggtccgca acgtcttccg ggagctggagatcctgcagg agatcgagca cgtcttcctg 300 gtgaacctct ggtactcctt ccaggacgaggaggacatgt tcatggtcgt ggacctgcta 360 ctgggcgggg acctgcgcta ccacctgcagcagaacgtgc agttctccga ggacacggtg 420 aggctgtaca tctgcgagat ggcactggctctggactacc tgcgcggcca gcacatcatc 480 cacagagatg tcaagcctga caacattctcctggatgaga gaggacatgc acacctgacc 540 gacttcaaca ttgccaccat catcaaggacggggagcggg cgacggcatt agcaggcacc 600 aagccgtaca tggctccgga gatcttccactcttttgtca acggcgggac cggctactcc 660 ttcgaggtgg actggtggtc ggtgggggtgatggcctatg agctgctgcg aggatggagg 720 ccctatgaca tccactccag caacgccgtggagtccctgg tgcagctgtt cagcaccgtg 780 agcgtccagt atgtccccac gtggtccaaggagatggtgg ccttgctgcg gaagctcctc 840 actgtgaacc ccgagcaccg gctctccagcctccaggacg tgcaggcagc cccggcgctg 900 gccggcgtgc tgtgggacca cctgagcgagaagagggtgg agccgggctt cgtgcccaac 960 aaaggccgtc tgcactgcga ccccacctttgagctggagg agatgatcct ggagtccagg 1020 cccctgcaca agaagaagaa gcgcctggccaagaacaagt cccgggacaa cagcagggac 1080 agctcccagt ccgagaatga ctatcttcaagactgcctcg atgccatcca gcaagacttc 1140 gtgattttta acagagaaaa gctgaagaggagccaggacc tcccgaggga gcctctcccc 1200 gcccctgagt ccagggatgc tgcggagcctgtggaggacg aggcggaacg ctccgccctg 1260 cccatgtgcg gccccatttg cccctcggccgggagcggct aggccgggac gcccgtggtc 1320 ctcacccctt gagctgcttt ggagactcggctgccagagg gagggccatg ggccgaggcc 1380 tggcattcac gttcccaccc agcctggctggcggtgccca cagtgccccg gacacatttc 1440 acacctcagg ctcgtggtgg tgcaggggacaagaggctgt gggtgcaggg gacacctgtg 1500 gagggcattt cccgtgggcc cccgagacccgcctagatgg aggaagcgct gctgggcgcc 1560 ctcttaccgc tcacggggag ctggggccatggatgggaca ggagtctttg tccctgctca 1620 gcccggaggc tgtgcacggc cctcgtcacaaggtgaccct tgcagcacag gccgcgggtg 1680 ccccaggctc ggctcagttc ttggaggtcaagggcatggg ttggggtagt gggtggggag 1740 gtgaatgttt tctagagatt caaactgctccagcaatttc tgtatagttt tcacctctga 1800 gaattacaat gtgagaaccg cacaaaaaaaaaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1860 aaaa 1864 2 369 PRT Homo sapiens 2Met Tyr Ala Met Lys Tyr Met Asn Lys Gln Gln Cys Ile Glu Arg Asp 1 5 1015 Glu Val Arg Asn Val Phe Arg Glu Leu Glu Ile Leu Gln Glu Ile Glu 20 2530 His Val Phe Leu Val Asn Leu Trp Tyr Ser Phe Gln Asp Glu Glu Asp 35 4045 Met Phe Met Val Val Asp Leu Leu Leu Gly Gly Asp Leu Arg Tyr His 50 5560 Leu Gln Gln Asn Val Gln Phe Ser Glu Asp Thr Val Arg Leu Tyr Ile 65 7075 80 Cys Glu Met Ala Leu Ala Leu Asp Tyr Leu Arg Gly Gln His Ile Ile 8590 95 His Arg Asp Val Lys Pro Asp Asn Ile Leu Leu Asp Glu Arg Gly His100 105 110 Ala His Leu Thr Asp Phe Asn Ile Ala Thr Ile Ile Lys Asp GlyGlu 115 120 125 Arg Ala Thr Ala Leu Ala Gly Thr Lys Pro Tyr Met Ala ProGlu Ile 130 135 140 Phe His Ser Phe Val Asn Gly Gly Thr Gly Tyr Ser PheGlu Val Asp 145 150 155 160 Trp Trp Ser Val Gly Val Met Ala Tyr Glu LeuLeu Arg Gly Trp Arg 165 170 175 Pro Tyr Asp Ile His Ser Ser Asn Ala ValGlu Ser Leu Val Gln Leu 180 185 190 Phe Ser Thr Val Ser Val Gln Tyr ValPro Thr Trp Ser Lys Glu Met 195 200 205 Val Ala Leu Leu Arg Lys Leu LeuThr Val Asn Pro Glu His Arg Leu 210 215 220 Ser Ser Leu Gln Asp Val GlnAla Ala Pro Ala Leu Ala Gly Val Leu 225 230 235 240 Trp Asp His Leu SerGlu Lys Arg Val Glu Pro Gly Phe Val Pro Asn 245 250 255 Lys Gly Arg LeuHis Cys Asp Pro Thr Phe Glu Leu Glu Glu Met Ile 260 265 270 Leu Glu SerArg Pro Leu His Lys Lys Lys Lys Arg Leu Ala Lys Asn 275 280 285 Lys SerArg Asp Asn Ser Arg Asp Ser Ser Gln Ser Glu Asn Asp Tyr 290 295 300 LeuGln Asp Cys Leu Asp Ala Ile Gln Gln Asp Phe Val Ile Phe Asn 305 310 315320 Arg Glu Lys Leu Lys Arg Ser Gln Asp Leu Pro Arg Glu Pro Leu Pro 325330 335 Ala Pro Glu Ser Arg Asp Ala Ala Glu Pro Val Glu Asp Glu Ala Glu340 345 350 Arg Ser Ala Leu Pro Met Cys Gly Pro Ile Cys Pro Ser Ala GlySer 355 360 365 Gly 3 25603 DNA Homo sapiens 3 gatggagccc aggccacatgcttgggaaag cgggtggtcc tgggtgagcc tgtctggcag 60 gagggtgagg tcctgtctggagtagacacc cggtctgtgt ccgccgcaca tgtgacaggg 120 cccagagggc acgggaagcccaggtgccct gtcccctggt gagtgggctg tgggggttga 180 gcaccccaag ggaaggcgtgttcttccaga gattcccccg taagagctga gctgcatcgt 240 gagcaggagg ggcagagggagcctggagag ggttgggtcg cctgggcagg cagctttctg 300 gggtggtgca caagcgacagaaagcctgag gcttgggacc ggctgaagac ggccttgggc 360 tgcgggtgct ggcgccgctccaggcagagg tgggctctgc agcccccccc cccccgccag 420 cttccccccc agcagcaagagccctggccc cagccagtgg ccccaagcca ggctcctgag 480 gggcagaggg tgagggccggattttccacc atatttgtct cacagcctgt ctggtcccag 540 ccccagggca acaaacagcctttctggagc agtttccaga cctgcagtgg ccgccttgag 600 cctgcagtga ccgtctgcaggaggccgcgg gtgctggggc tggcgcagga aagcaccgtt 660 gcttctgcgc ctgtgcagagtgaggctggg gcttccatcc cgggcacggg actcctcggc 720 ctcctgcggc cgtgtgcatgggaggaaggc cgtgctgccg agccacgcga ctctgccccg 780 ttggcagtgg gaagcggcaggagggggtcc tgccaggggc agccaggggc tgctgcagct 840 tacgctcact gtcattctgaaaccctcaac tggctttcaa aataacaatt taaaaaatgg 900 tcataggaaa tgcaggaagttcaggagaaa tcccgccccg ccccgccctc ccaaggcggc 960 ctcgttcaag cttagtctccgtctgttctc gggctgcctg cagctgcccc cgctcctcag 1020 caggtgtggc cgcgtgttcaggagccccca tcagcacacg cgcctctggg cagcccccaa 1080 cacaaggctc ttcctgtcccttcaggctca gctttcccct cccaccgggc agggaggtgc 1140 tgaggccacg cctgttgtcagcttcctgga gaggccatac ttagagccca tggtgccagg 1200 ccaagccact gtctcccgcaccacagtgct gcagccggat ccacccaggc caccgcttgg 1260 caccatcaga catgctttcttagtttgggc cagcccggtg ctctgtgcac agtgtgactc 1320 ccagtggccc cttgcgggagggagggtcac cgctttgctt cacaaaagga ctgagtctca 1380 gggaggggtc tccagtaagggccccggagc caggatgtga ctgagacagg tgcctccagg 1440 gccacacact aaccagtacaggaacctctg ggggcagaat catggcctcc aaaacaccca 1500 catccaaatc ccagaacaaacatgttacca ttcgtggcag agaggaatga agattgcaga 1560 ttgaattaag gttgttaatcatctgactta atttttttag agacagggtc tcgccctgtc 1620 acccaggctg agttcagtggtacagtcgtg gctcactgca gcctcaacct ctcaggctca 1680 agcaatcctc ccttctcagtctcctgagta gctgggacta cgggtgtgtg ccaccacgct 1740 ttccgggcat gcagccaggagcccagggcc atctgtggcc caccttgaga tccagaatca 1800 tccatttcct ccaggccccctgctgggctc caactccttg aggaccagag agcagaggtt 1860 gtggaaggcc ttggaaacgggtctggatta cctgtcctgg gaaggtctct cccaacctga 1920 gtgtcagaca ggggttagctctgctgctca caattttgtg ccttaattcc tggcttccct 1980 ttggggatct tcatcctcaattctgattga catccttggc cacaagggac ccccctgctc 2040 attgatgctt ctcacccgtcacctcactct catcctcact gctaagcaat tagccgtgtg 2100 tttgcggcat cagtgttgacaccgatgatc catgctcaga gggtacaggc ctgaagagct 2160 atgtggggac tggcgccccggagggggtcc cgctgtggtg gcagcggtgg cccccaagcc 2220 ccacgctcac tctgtgtgtctccttgcagg acagggtgag gggccttggc ctcacggtgt 2280 tgagacggga gtcggtctctggagtgtgga gtgatgtgcg tccagggtga agctgcagcc 2340 atggttgatg aggcttcctgagcgcagggc tctgtgctca gtgggttttc gcattcattc 2400 cccagtaccc ctccgggctgcttgctgctc caagccctgg aaaaggatgt tggggtttag 2460 gaaggcaaaa ctccatgcccaggtcgtggc tggtgagggg cgctccccgc acaaccagcc 2520 actgcttggc tccaactcacgcctggatgc tgttaggctg gaccctgtct gtttgcagat 2580 agcgcctgtt gacagatgtgtcctgctgca gacttgaaac gcaggactga gtctcaggga 2640 ggggtcttca ctaacagccatggagccagg atgtcactga gacaggtgcc gccagggcca 2700 cacactaacc agtacaggaacctctggggg gcagaatcac ggcctccaaa acacccacat 2760 cttaatcccc agagcaaatatgttaccata tgtggcagag aggaatgcac ctgccgcgtc 2820 taggaggcag aggggctgcggcgcgtcccc aggtgtcccc ttgtgtcctg acgatgcgtc 2880 cccaggcatc cccatgcatttccaggtgtc cccacgtgtc cccaggcgtc ctcaggcgtc 2940 ctgccgaaca gccctgtgccctccaggtgt gcattgtgca gaagcgggac acggagaaga 3000 tgtacgccat gaagtacatgaacaagcagc agtgcatcga gcgcgacgag gtccgcaacg 3060 tcttccggga gctggagatcctgcaggaga tcgagcacgt cttcctggtg aacctctggt 3120 gagcctgcca tggcctcgctgcagaaagac ttaccgtcct gaagccggga aggcactggc 3180 tatcctctcc tgcccttggtgctccttggc cctcgtgcct cggagatgcc tctgccaccc 3240 acaggccctc tctagccctcctcatctatc ccccttcctg tgccccaggc ctggcagtgg 3300 cccaggtggc catgacatgctggggttggt taatgcagtg tctcttctga gcctgccgga 3360 agaccagggc ttccctacaatggagatgtg ctcccatgga gtctctggca ctagtcagag 3420 agggagagag tttagggactgaaaaactca ccactgtcat caccatcacc atcaccacca 3480 tcatcgccat caccaccaccatcaccactg caaccatcat cactctcatc accatcatca 3540 ctataatcaa cacaatcactattgtcacca ccattaccac caccaccacg accacaatca 3600 ctgttatcac catcaccaccaccctcagtc ctcactaccg tcatcctcac catcaccgtc 3660 accaccacca ccatcactgccatcgtcaac accatggatg ctggttgtta aatgccagct 3720 ctttgcccaa cactgtcaagagtggtacct acacggcctc attttctgta acaaccctcc 3780 gaggcaaatg tctgtatccccattttaccg aagaggaggc cgggcagcct gaagcacccg 3840 gagctggcac tgtagctctgctctgcattt gccactccca ggtgcctctg gccccagctg 3900 ggccacctcc agcacagggtggtgtgtctt tcctcaggat ctgggctcag agctgctctg 3960 ggctggggtg caatcagtgccttgggcagg cccctcctcc tgggaatgcc tggtggctga 4020 tgctggggtg gggctgtggtccttaggggg agtgtgtcag ctgtgggagc agccatgact 4080 ggctccccag ctgtgcgcacaacaggcctt ccatcggtgc ccacaggtac tccttccagg 4140 acgaggagga catgttcatggtcgtggacc tgctactggg cggggacctg cgctaccacc 4200 tgcagcagaa cgtgcagttctccgaggaca cggtgaggct gtacatctgc gagatggcac 4260 tggctctgga ctacctgcgcggccagcaca tcatccacag gtgtgtgcgt ggcagacggc 4320 gcaggtacct gctgaggtgggcggggctga agcagcctta ggtcaggctg ccggcacggc 4380 ggccgtactc cctcagagcgggtctagctc ctctgcccca cccttgcctg agtgcctgcc 4440 cccagctgtg gcacctgtgccgaccaggtc agccccatag ctgtgtgcct ggtgtccatc 4500 tggggggacc tcgtcccagcagccccagct gagactgggc acagtgggct gttagccctg 4560 gtggacagac caccaggctgggtcacagca ggtggccttc acctggtcca tttaactgaa 4620 gactcctgtt tgcccatccaccacatccca gggaatccaa actaatttta acattagctt 4680 aaagcagatg aaattaggaagcagagctgg tgtgatggct ctgaaaataa aatttaaaaa 4740 aagaaaatag gaagcagattatgaaggaag tgaaattggg aagcagaaat taggctgaaa 4800 ttccgcagca atggaacaaaatgaaaatat ctgtgaggta tattttaaag tcgaatggac 4860 tggtgtttgc atttctgctcttggggactc ggatgtctga ttatgaccta ggcaccagtc 4920 actgagcact ggctgtgtacctggaaaagt tgggacaaag caagagccga ggtggcttgg 4980 tctcctagag gccgagtcttggagggggag ggcagaccct gccagcaatt gctctcgtcc 5040 tctggggctc caggccccctcccagcatct ggtgccaggt gtgtgctgct gcccagatgc 5100 cacagggaac gaagtggctgacttcatcgc ctctgccccc acgcaggggt gtgaggtcct 5160 agcatcatcc aaggaccaagtcaagctccc aggcctctgc ctcgagtggg ttggtgggat 5220 gtcctgggga ctccagggattgtgacagag attccagggc agaaacaggg cagattccca 5280 actcaccttc ccactttctgctctttctag agatgtcaag cctgacaaca ttctcctgga 5340 tgagagaggt gtgtggggttgggtgtgggc agcccaggtg ggtggtggca gggatgggcc 5400 tgtcagggga ggaggatcctgcacgcaagg atgcatctct ggtcctggga cagccacacc 5460 tgacccctct ctgcacaggacatgcacacc tgaccgactt caacattgcc accatcatca 5520 aggacgggga gcgggcgacggcattagcag gcaccaagcc gtacatgggt gagcccgagc 5580 tggggttcca gatgggagctggcttcctcc aggtgggaag gacaagacct cggtggcttc 5640 tctgtcccac cctggaggcagcctggtctc gggatgtggc ctcaaggtgc cggccctgtg 5700 cccacgggtc cgggctgtgaccccgtggca gctgtttttc cttctttctg tcggaaagct 5760 ccggagatct tccactcttttgtcaacggc gggaccggct actccttcga ggtggactgg 5820 tggtcggtgg gggtgatggcctatgagctg ctgcgaggat gggtatggac cccctgcagc 5880 ccccgggctt ggctgccaggcccctgctct ctgcccccac cagtgctggg gagggggtgg 5940 ctgccccagt gcccaggtgcgcagggatgt ctccactgtg tctgaggagt cacgctttta 6000 tcgaagtgtg tagttggtgatggaatgcct gagcaggagg aggaaggaca gactcactgt 6060 ggtttcccgg ggccgctgctggtgcctgca ggccagcctc tgtgggggtg gacaaggctg 6120 agaactggcc agcaggggtgctgcctcgga actttccaca aaaagtttct tttggggccc 6180 tgtgctctta cccttgtggccacggcgagg ccagtcctgg agaccgggag gctgggggtc 6240 ctcttgtgga ccgtacccctcagccctgca caggacccca cctctgagga agccagctcc 6300 ctcctggccc tctggggctgatctacctgg acccaggccc cctgggatcc cagccagatg 6360 ggcgcagcag ccagggcgcaggacccaggc gtaagcttta tctcacccag gctcctccgc 6420 ggcaggtgga ggccaggctgtgctcagagc tgtgcctgca cttggggtgg ggggaggggg 6480 tcctctcagg gcgatggcacctgtgtctgg cattgttctg ggtgtcctgg gggccaggag 6540 gacctgccca gcactgcctccctgtctcca gaggccctat gacatccact ccagcaacgc 6600 cgtggagtcc ctggtgcagctgttcagcac cgtgagcgtc cagtatgtcc ccacgtggtc 6660 caaggagatg gtggccttgctgcggaaggt gagcccccat ccctgagcct cctcaccctc 6720 cgagcaccca cctccctccctcacttacct gcggctcggg acaccccctc cagtgcacag 6780 ttagtgccgc ttcctggcaggcacagatcc cttcactgca acctgtgggg gcctccgcag 6840 atggcagccc caagccccaggaagcgagct ggtggcaggc tctgtggccc tctcatggcg 6900 agccctacca ggtcactggtgtcctggtga tcccctgagc tgcgtctcca ggcaccctca 6960 cagcaggctt gtgcgcccgccctgccggtc accacagagg agccctgagc cacgtcccct 7020 tgtcccacgt attggaaggagggtgtcggg tgggtgggtt caagcccatg ctatctccgg 7080 gaccctttgc cccagtgcctcctggggaag gtgggcagcc actgcccact gcaaacacct 7140 ctcaggggac atcagcctggcagggacacg ggcaggggtg tggccatcag tagtgcctcc 7200 catttgtgat ctggtgctaggctggggctg tgccttgact gggctgcaca gctccctgtt 7260 ccgagcctcg cattaacaccatagggggtt cggagtcaga gccaggccca ggcaggacag 7320 ggaggggagt gagtgtgccacacgggcccg gctgcctccc ggcccccgtg tctcaggcag 7380 gtggggcctc ctgccctggaattgtagcca agcagcctaa agccttgggg aggccttgcc 7440 tgccggggcc tctccccagccccgagagtc tcttaactct gctgtagccc catgaagctc 7500 agtcacacct gcccaggtggctcacaaggt ggcactgggc tagagagggc ctgcgtgggg 7560 actggggatg acccacacgcccaagcccag gtctgggaaa cctcgcacgg ggtctgggtc 7620 tgcggcattt tccctggaaaggcgggaggt gccagcgctg ggatgttgct tcccaggcca 7680 tgcatggctg ccccgggctcatctggcctg tggaggtccc atgattcggt gaaggaagtg 7740 gctctgggat agttactgtgaggccagcca tgtgccgagt gttagccgct agccgggcct 7800 cggctgccac ctcctggcaaatcccagcag agccttccct gcagatccct ctgctgtcct 7860 ctggcgccag gggtttaggtagcagcactg agaacaggcg tcccttgggc cacatgctga 7920 gccagccacg gtgctttgcctgatgtcggc cgtcggcacc acccttcctc gcgtggccct 7980 gaggttcctg aattctgaacctgaggcttg gtgggaccct cctcaaggtg ccctggcctg 8040 ggggtggcgg gctattccgtgctggtgggc tgtgggccct ggaccctctg actcatgcct 8100 ggttgcagct cctcactgtgaaccccgagc accggctctc cagcctccag gacgtgcagg 8160 cagccccggc gctggccggcgtgctgtggg accacctgag cgagaagagg gtggagccgg 8220 gcttcgtgcc caacgtaagcctgtgggcgg ctcaggtggg gggccctggg gatggatgtg 8280 gcgtcctcca cgggccggggctcagcaccc atccctctgt agaaaggccg tctgcactgc 8340 gaccccacct ttgagctggaggagatgatc ctggagtcca ggcccctgca caagaagaag 8400 aagcgcctgg ccaagaacaagtcccgggac aacagcaggg acagctccca gtccgtgagt 8460 gccagggcag gctcagggcgcggcggcggg ctgggcttgg ggctcctctc taccaccgag 8520 caaggtgtgt ggggacccctgacagtgcac acgtctcgga agtccagcag accgtttcct 8580 gaagtcctga gaaggccagagacctccctt ctgcctttcc cagcccccac ctcgctcctt 8640 atgaagcagg tgggcagggacaaccagggc tggggttatg agtgcacggg gatggccatg 8700 tgaagccttc gtgcttgcccaggtgtgctg gtgttggttg tgtgtgcggg gacggctatg 8760 tgaagccctc acactcgcccaggtgcgtcg gcatcaggta tgtgtgccgg gacagccatg 8820 tgaagccctc acactcacccaggtgcgtcg gcatcagttg tgtgtgtggg gacggccatg 8880 tgaagccctc acactcgcccaggtgtgctg gctttggttg tgtgtgcagg gatggccaca 8940 tgaagccctc acactcgcccaggtgcgtca gcatcaggtg tgtgtgcggg gacggccatg 9000 tgaagccctc acactcgcccaggtgcgttg atgttgtgtg tgcagggatg gccatgtgaa 9060 gccctcacac tcacccaggtgcgttgatgt cagttgtgtg tgcagggaca gccatgtgaa 9120 gccctcagac tagcccaggtgtgtcggtgt cagttgtgtg tgtggggatg gccacgtgaa 9180 gccctcacac ttgcccaggtgcgttgatat tagttgtgtg tgcagggatg gccacgtgaa 9240 gccctcacac tcacccaggtgcgttgatgt cagttgtgag tgtgcgcagg gatggccaca 9300 tgaagccctc agactcgcccaggtgtgctg gctttggttg tgtgtgcagg gacggccatg 9360 tgaagccctc acactcgcccaggtgcgtca gcatcagttg tgtgtgtggg gacggccatg 9420 tgaagccctc acactcacccaggtgtgtcg acatcagttg tgtgtggggg gacggccatc 9480 tgaagccctc acactcacccaggtgtgtcg gtgtcagttg tgtgtgcggg gatggccacg 9540 tgaagccctc acacttgcccaggtgcgttg atattagttg tgtgtgcagg gatggccacg 9600 tgaagccctc acactcacccaggtgcgttg atgtcagttg tgagtgtgtg cagggatggc 9660 cacgtgaagc cctcagactagcccaggtgt gctggctttg gttgtgtgtg cagggacggc 9720 catgtgaagc cctcacactcgcccaggtgc gtcagcatca gttgtgtgtg tggggacggc 9780 catgtgaagc cctcacactcgcccaggtgc gtcagcatca gttgtgtgtg tggggatggc 9840 cacgtgaagc cctcagactagcccaggtgc gtcggcatca ggtgtgtgtg ccgggacagc 9900 cacgtgaagc cctcacactcgcccaggtgt gccggctttg gttgtgtgtg cggggacggc 9960 cacgtgaagc cctcatgctcactcaggcat gctggtattc tggggctgcc aggacaggtg 10020 accacgaatc aggtggttgaagaacagcaa tgcgtctctc tgagaggatc tgagtcgtaa 10080 tgaaatggtc tccttcacagccggctgtgc gtgaactact ctgtctcctg cagctcccct 10140 gtcttgataa ttggctgtctaggcagcggg taaggtgaac cccttgggca gttatgtgat 10200 gatctcagtt tctgtaaaccggaagtccag gcatggtgca gctctgttcc ctgcttcggg 10260 gtctcaccag aatgtgagctaacattgagg tcgtggcctt gtcaggtgca gctctgtttc 10320 ctgcttctgg gtctccccagagtgtgagct aatattgtct gaggtcgtgg tctcatcagg 10380 gatttgacag gtgctgtggttgaaatgttt cccttaaaac tcgtgttgga atttgcttcc 10440 tattgtgatg gtggtagagatgggactttt gggggctgat ggggccacgt aggttcttcc 10500 agcatggatg gggttaatgctgttgtagaa gggtgacttt agtcctcttt tgagtctttg 10560 atcctctgct atgtgaggacgtggtgttcc caatgtggac gtggttcgtg ttccatgtga 10620 atgtgatatt cacaatagagcatcaacagg ctccctttta atcagcagat ttaaaaagaa 10680 atgtgttgtc tcatggcttggaggcctgag tccaaagtta agatgtcagc aaagccgtgc 10740 cccctctgaa ggctctccggggaggaaaac cagtccttgc ccctctaccc tccggtagag 10800 gctgccttgg cctagacgcatccccccagc ccctgcttcg ctgccgcgtg gggtcggcct 10860 gtgtgtgcgt ctccatctcctcccctcttc tcataaggac accaggcatt ggatttaggg 10920 cccaccctga tccagtatggccccatctta tcttgatgat atctgcaaag acctcacttc 10980 caaatgaggt cacattcacaggtacccagg attagaattt gagtgtgtca tttttgggga 11040 cacagtttgg cccataccaccaggatgtgg ctgatattca ccaaggagta gctatggttg 11100 tgtgttgatg tcagggtgacggtgatgacc ctgggtccct cggtggtccc cttgcccctg 11160 agtctgcctg agcctgtggtggatgtcctg ggaaactctt gtgcctcagc ccccgtgcag 11220 cctcctcaga cctggtgggccctgtgttgc tcctgggcag aagacgggtg tcagtcccct 11280 cctcaccatg atgtggggggcaggggtggg gtcatgccct gggtgccctg attttggggg 11340 gaacacggcc ccccagtgggtcaggctccc atcctcgccc ctcctccagg acggctgccg 11400 gcagccctgg gtgtcctcaggcagaattgc tggtggagag ctgctgtctg ccaggtggcc 11460 actgtgaggc actgctgagagccacaggat ggttggaagg ttctcggggt tgggggttct 11520 ttggcattgc ccccattggatgtttaagtt ttccctacca gagcatgtcc agagccaggg 11580 ctctggggtg tagaaacaggcccaggatga gttaggaacc ctcatgggag actcagggat 11640 ggacagtgtg cagagcccagctggccatgc tgagttccca ggaggctctg gctgggaaca 11700 ggtaaggcca ggcacctgtgagcgggagga gctcggcttt gtcttgggtt ggttgtgtgg 11760 agatgttttg gcttgagggtaggaggtgtt ctgaaaggaa agcatcactc caaaaaaaaa 11820 gtcccactgt taaccttgaggctgagagag gtttttggaa acagctttat tttgatataa 11880 ttcacattcc atgcaatatacagtgcatcc atgtaaagca tataattcca tggtttttaa 11940 tatagtcaca gggctgtgcattctccacca caatctgatt ttagaacctt ttcatgtaat 12000 gtaagagaaa gaccccacctattagcagtc atgccccatt cccctcttct cccctcccct 12060 ggcagccacg aagctactttccgtctctgt agggttgcct gttgtgggcg tttcatggaa 12120 gtggagttac acactatgtggtctttgcag ctggcttctt tcacttcgca ggatgctttt 12180 gaggcccgtc cacgttgtagcctgtcagtg cttcattcct gttgatggct gagtaatatt 12240 ccacatatgt atcacctttcctttatccag tcatcagttg atgagtattt gttctttcca 12300 cttgttaaca tttttcattatcatgaataa tgctgctgtg aacattcaca tacaagtctt 12360 tgtgtggata tgtacttttatttttggggg gcacatacct aggcatgaac ccgctgggtc 12420 atatgtgact ctgtgcttcatgtttggagg aaacacctac ccttttctac agcaggtgtg 12480 ccattttatg cccctaccagcagtgtgtga gggttctaat ttctccatat atttaccaag 12540 tcctgttatt gtctggtttatttttttaaa aatcatagtt atcttagtgt gcagtgattg 12600 tgtggttatg gtttgcatttctctgatgat attgaacatc ttttgaggtg ttttatcagg 12660 cattgtgtct agagaaatgtctatccaaat gttttaaaat ttttattgtc ttttttttag 12720 tctactctga caatatattttaattggcat atttatttta ctttattttt ttttagagac 12780 agggtcttgc tttattaccaaaactggagt gcagtggtgc aatgaaggct cacttcagcc 12840 ttgacttcct gggctcaagtgatcctccct gccccagctg ccagaatggc tggaactgtg 12900 ggtgtgcacc accacacctggctcatttga aaaaaatttt gttgtagaga cagggtctca 12960 ctatgttgtc caggttggtctcaaactctt ggagtcctcc cacctcagcc tcccaaaatg 13020 ctgggattac agatgtgagccactgtgcct gacctaattg gtgtatttta gaccattcac 13080 atttaaagcg accagggaggctgaggcaag aggactgctt gagtccagga gtttgagacc 13140 agcctgggca acaaggtgagacccatctgt attagtctat tttcacactg ctaataaagg 13200 catacctgag tctgggtaatttatacagga aaaaggttta atggatttac agttccacat 13260 ggctggggag gcctcacagtcgtggaaggc aaggaggaga aagtcacatc ttacatggat 13320 ggcggcaggc aaagagagagcttgttcagg gaaacttttg tttttaaaac catcggatct 13380 catgagactc attcactatcatgacaacag cacaggaaag acccgccccc ataattcaat 13440 cacctcccac caggttcctcccacaacatg tgggaattgt gggagtcaca attcaagctg 13500 agattgggat ggggacagagccaaaccata tcattctgcc ccagcccctc caaaatctca 13560 tgtcctcaca tttcagaaccaatcatgcct tcccaacagt cccccatagt cttattttgg 13620 cattaactca aaagtccacagtccaatgtc tcatctgaga caaggtaagt cccttctgcc 13680 tatgagtctg taaaatcaaaagcaagtgac ttcctagata caataggggt acaggcattg 13740 ggtaaattca gccattccaaatgggagaaa ttggccaaaa caaaggggct acaggcccca 13800 tgcaagtctg aaatccagcaggcctgtcaa atcttaaagc ttcaaaatga acatctttga 13860 ctctatctct cacatccaggtcatgctgat gcaagagttg ggttcccatg gtcttaggca 13920 gctctgccct tgtggctctgcagagtacag ccttcctccc ggctgctttc gtgggctggc 13980 attgagtgtc tgtggcttttccaggtgcat ggtgcaagct gttggtggat ataccattct 14040 ggggtgtaga ggatggtggccctcttctca gagctccact aggcagtgcc ccagtgggga 14100 ctttgtatag gggcaccaaccccacatttc ccttctgcat tgccctagca gaggttctcc 14160 atgagggccc cacccctgcaacaaacttct gcctggacat ccaggtgttt ccatacatcc 14220 tctgaaatgc aggcagaggctcccaaacct caattcttga cttctgtgca cctgcgggct 14280 caacaccaca tggaagctgccacagcttgg ggcttgtacc ctctgaagcc acagcctgag 14340 ctgtaccttg gcccctttcagtcatggctg gagcagctgg gatgcagtgc agcaagtccc 14400 tagactgcac acagcagagggaccctggac ctcgcccatg aaaccatttt ttcctcctag 14460 gcctctgagt ctgtgatggtaggggctgcc gcaaaggtct gtggcatgcc ctggagacat 14520 tttccccatt gtcttggtgattaatattca gttccttgtt gcttatgcaa atttctcctc 14580 agaaaatggg gttttcttttttttctctct tttttttttt tttttgagac agtcttgctc 14640 tgtcacccag gctggagtgcagtggtgcaa tggcggctca ttgccactgc aacctccgct 14700 tcctaagttc aagtgattctcctgtctcag cctcccaagt agctgggatt acaggcacgc 14760 accaccacac ccagctaatttttgtatttt tagtagagaa gggtttcacc atgttggcca 14820 ggctggtctt gaactcctgacctcaggtga tctgcctgcc ttaacctccc aaagtgctag 14880 gattacaggc gtgagccaccgtgcccagcc aggagtttct tttctattgc attgtcaggt 14940 tgcaaatttt ttgaacttttatgctgtttc ctttttaaaa tggaatgcct ttaacagcac 15000 ccaagtcacc tcttgaatgctttgctgctt agaaatttct tctgccacat accctaaatc 15060 atctctcaaa ttcagagttccacaaatctc tagggcaggg gcaaaatgct gccagtctct 15120 ttgcttaaag cataacaagagccacctttg ctgtagttcc caacaagttc ctcatctcca 15180 tctgagacca actcagcctggacttcattg tccatatcat tatcagcatt ttggtcaaag 15240 ccattcaaca agtctctaggaagttccaaa ctttcccaca ttttcctgtc ttcttctgag 15300 ccctccagat ggttccagcctctgcctatt acccagttct aaaaagttgc ttccacattt 15360 tcaggtatca tttcagcagcgccctacttt actggtacca atttactgta ttagtctgtt 15420 ctcacgctgc taataaagacatatccgaga ctgggaaatt tatacaggaa aaaggtttaa 15480 tggacttaca gttccacatggctggggagg cctcacaatc atggcggaag gcaaggagga 15540 gcaagtcaca tcttacatggatggcagaga gagcttgtgc agggaaactt ttgtttttaa 15600 aaccatcaga tctcatgagactcattcact atcatgacaa cagcacagga aagacccgcc 15660 cccataattc aatcacctcccactgggttc ctcccatgac acacgggaat tgtgggagtc 15720 acaattcaag ctgagattggggtggggaga cagccaaacc ttatcaccag ctctataaaa 15780 gacaaaaaaa ttaggcaggcataacagtgc atgcctgtag ttccagtgat gtgagaggat 15840 tgcttgagtc caggagtttgagaccaggct gggcaacatg gcgagaccct gtctctacaa 15900 aaaaaaatta tctgggtgtggtgggataca cctgtgatac cagctacgca ggaggctgag 15960 gcagtaggat tgcttgagcccaggagttca aggctgcagt aagctatgat catgcccctg 16020 cactccagtc tgggtaacagagagacacgc tgtcttgtaa ataaataagt ggtcatttat 16080 atagttcaat atgatatctaccttatttgt aactgtagtc tatttattgg tcttcctttt 16140 tccctatttt tctgccttttctggttttaa ttaagcattt tatattattc tagtttatct 16200 cctctcctgg cctgttaattatacttcttt ttgaaatatt tttagtggtt ggcctggaca 16260 ttgcagtata cctttaccatacagtctacc ttcacctgac actctgcccc ctcatgtgca 16320 gtgggatgcc ttgtgacggcacctctcgtg caatcctcct gttcctgatg acattgctgt 16380 cattcatttc atttatctgtatgctataat tgctcattac attgttacta ctgttatttt 16440 aaacagttat cttttggatcaattaagaaa aattaaaaat ttcattttac ctctattcat 16500 tcctcctcta aagtgcttcctttctttatg cagacccaag ttgctgacct aaatcatttt 16560 cctttcccct gaggaacttcgtttaacatg tcttatagga caggtccaac agagatgaat 16620 tccttccctt tttgtttgtccaaaaaagtc tttactttca cctttaaaga ataatttcac 16680 tggatataga attctagattggtaggtttt ttactttcaa cactttaaat atttcactcc 16740 gctctcttct tgcttatgtgatttctaaca ggcagtctgc tctaattctt tttctgtaag 16800 tagaattcct cgccaccccccacccccagc tcatttcaag attttctctg tctttggttt 16860 tctgcaattt gaataatgatatgcctaggt acagattttt ttttaatatt cattctgctt 16920 ggtgttctct gagcttcttagatctgtggt ttggtgtctg tcattaattt cagaaaattc 16980 ccagtcatta ttatttcaaatattctgttc tctttctctt ctcctgctgg aaatccaatt 17040 atgtgtgtgt ggtgccgtttgaaattgtcc cacagctctt ggatattctg ttcatttttt 17100 tcactctttt ttttttctctttgcatttca gttcgagaag tttctgttga catttcttca 17160 agctcataga tccttcctcatctggatcca gtctactgag gaaccatcaa aggcatcctc 17220 atttctgtta cagtgttttctacttccagc atttcctttt gatccctcct tggagtttcc 17280 atctctctac ttacattgcccttctgttcc ccatctgttc ttgcctgctt ttcccctgga 17340 gcccttgcca tattaatcacagttatttca aatttcctgt ttgataattc caacatgggt 17400 gccatatctg gatctgttctaatgtgtgca ttgtctcttc acactctatt tttttcctta 17460 agaccttgta gtctgccttgtaatgtttgt tgaaagctgg acgtgatgta tctggtaata 17520 agagctgaag tagatgggcctttagtgtga ggtttatgta atctggccag gtttggggca 17580 gggtttaaag tctgctgtagctgtgggtac cagaggcttc acatttgttt tcatttcccg 17640 ggttgccctt gggcttgcctaaatcctcct cctcagagag agtctgcgtc ttgtggccct 17700 ctctgctgga atccctgtcacactgcggag gccctgtggg tgtgttggga agatggtggg 17760 gagggaactg ttccacagtctgtgaccaaa tctcagtctt gggggcctgt gccccttcac 17820 agttgttgat ctgcttttcccctcccctta ggtgagacag gctaaagcgg ggtacaggct 17880 gaaaaacagt cctcccccaagtgagataag cctttccttt ggagagcaaa ttccatttgc 17940 tgtggagaat gctctgggtgtatttcacag tggtgactgt ccccatccca tgccagagcc 18000 aggaagggat catccttggcttcattaaga cctggcaggg ttcctggagg ggaaacccac 18060 aaaacgttgg gggcctcataagaccgcagc cgcaggagtt ctgcacacgg ccccggccgc 18120 tccccagagc tgcccaggaggtgttcccgc acaccatcag ttctgctcca ggccagcagg 18180 tctcagctgt gactttgctcacctgtctct ccagacttgg gggtcgccgc agcgctttga 18240 cctcagctct ctgatgggtccaggaaaagt aattgatttt cattagttca gcatttttcc 18300 tgtcgcaggg atgggagtgaggccttccac gttgtttgcc gcagagcaga agccagaagt 18360 ccttgctcgt ctctgtgtttcttcgtcgga gtcatgtctt ttatttcctg aacgcaggtg 18420 tgtggtcaga caggagattggcaggtattt cctcctgttc catgggctat cttttctctt 18480 tcttgagggc atcatttgcagcagggaagt cttggacctt gacgttgtcc gatgtggctg 18540 tttcctcctt ggctgctctgctttgtctaa ggatccatca cctcagctga ggccacaggg 18600 atttactccc acactttcttctaagtcttg tatagtttca gctcttgcgt ttagctgtgt 18660 gattcatttt gggttaatttttatgaacaa tgtcaggtga gggtccagct tcattctctc 18720 atcggtggat atctgactgtcctagcacca tttgctgaag agaggattct ttccccattg 18780 aattgctttt gacatcataccttgtttttt gacttgccgt tttatcccat tggtccagac 18840 gtctgccctg cgccgggactgcaccatctt gataactgta gctttgtagc aagctttcaa 18900 atcaggatct gtgaatcccccagttttgtt ctttttggac attatttgac tattctgggt 18960 ccctggcatt tccactgaatgctgaggggt ctgacagttg catctgagct gccaagcagg 19020 tttgtggcgg tgctagggactgaagcctgc tccatttccc aggcccctcc tcgctgtggg 19080 tgacatctgg ggtccgaggctgtgtctcag catgtgtgaa ggtgccacgg gtgccctgag 19140 atggggattc ctggtccagttactcagaaa gtgcatccag gagagacccc cgcccttctc 19200 gggatgggag atgccagcagagcttggctt tcaagcagaa atctggaaac cctgtgggga 19260 gtggcttcgg acttcaggggacctggagcg tcacttgtgg ttcaagaggt ccctgccctg 19320 agggagctgg gctctcaaacacccacacca gtcagtcatg ggcgaagggc ccctctgtgg 19380 ccttctgact gtgtgtgtgctggcaaaggg ttccagccac ccaaggagga ggcaggggct 19440 gtcagaggaa gagcagggcagatccaggcc aggcacagac cctgccatgg ggtactgctg 19500 gcggcgctac acacaccttgggaagggagt ccctgtggaa aggggtcgtg gtcacacatc 19560 taggtgacac agcccggcttgggcgctgct cagagccacc ccttccagat ggttctggag 19620 cagctcctca ggcttctggtggcctctctg cctaggaaaa catggctgtg gacgttgcag 19680 gatgaccaac agcccctgccactgggctgc acacagggcc acgacgggcg ctcatgttct 19740 acatcactgg cgcccaccccagcccctccc accttgtgtc gctgtgaatc gcaggatccc 19800 agcggctcag tcggaccctcattcctgagt agtctgagcc ttaggtcacc gtcaccttct 19860 caggccggcc cgagtttgcagacttgtctg tctatatcag ggttagacca gagagtgctg 19920 agacacagca gatcacccagcctgtcctct tcttgatgac taaggacagg tcccctgcca 19980 ggatcgtgac tcctttaggggaggccacag tgacagggca aagcctggag ggagagagcc 20040 acatggagag gagagggctgcccgcagaga gcgtgggagt ctgccggctt cttctcgagt 20100 ccttggcaag gtgctggccgctcacaccgt gtacgtgtgg ggaacgccca ggaccagggt 20160 gacaccacca ggaggagcgggcgggacagt ccccactcag ggctaggaag agagagtcca 20220 cattcccgct gccagggtgaaaccctcaca ccaccacaga tccaggagag acacggaggg 20280 cactgcctcg gggtggggaacgtgagctgc tccccaaacc caagaaatgt gttgagccct 20340 atgcttcctt cctcgtgggaagaggcgcaa ggtgagaccg cttgtccttt atcttggcgg 20400 gcacgtccga gtgtgacgcacgtcagcaaa tccctgaacc gttcatcgga gaacagcctt 20460 ctgcatctcc cacactctgttcgtgggttt acagggtgtc cagagtactt gccacttggc 20520 aatcagcatt aataggatccacagggccag gcatggtggc tcacgcctgt aatctcagca 20580 ctttgggagg ccgaagcaggcggagcacct gaggtcagga gttcaagact ggcgtggcca 20640 acatggtgaa accccgtctctacaaaaatt agctgggtat ggtggtgggc acctgtaatc 20700 ccagctactt gggaggctgaggcagcagaa tcacttgaac ctgggaggcg gagttggcag 20760 tgagccaaga tcacgccactgcactctagt ctgggtgaca gagcgagacc aaaaatagga 20820 tccatggata gcaggcaagagtgtccaggt gttcgaggca cagacgacac tgtgacaggg 20880 aagagtcccc ttagccctggctggggccgt gaaagcatgc tgttgtccgt ctcgggtgaa 20940 cgcagactgt tgtcacgcattgcataacga tgcttccgtc actggccaat cgcatggggg 21000 ggtggtcccg taagatggtaacactgggtt ttgctgtacg ttttgtatgt ctagataggg 21060 ttgagcgttc tggtgtgtacccactcacac gtcccctccg accttcagag cccagctccc 21120 tccctcccag ggccttggctgtgacgtggg tgacttccta tggatctgag gttctgtggt 21180 cctcacaagt gggcatcctctggcctcagc tgcaggaggt gggggccctt ttaatgccac 21240 ccgaggcctg cgactccctgcacttttcac tgtgacttgg ctcatctggg tctgtcattt 21300 gctcacgcgt tggtagtgaccaacgtcacc atccaagttc acggtcacca taatgatgct 21360 ttccccacac catgccagcgctgagcggcc agcaccccct ccaccccacc cacgccctcc 21420 cccgacccct gcgtcctgggaagtggtcct gctgcctgaa ggaagtagtc ctgcctgccc 21480 atcacacacc agtaagggtgggtcctgcca ggggcagcct ccgtccacaa gcttgccctg 21540 aggacctgct tctaagacagccctggttcc aggattctct gggcagggcc ccagaagcag 21600 gcctgggaca ggtgtgtgtgtgctgtgatg aggggctggg agaacccggt atgagacggg 21660 aaaggcccgg caagggagtggtttccagca aagtcccgca gagaacagct tctgcctggt 21720 cctgcaggcc ccacggagcaagtccaagcc atccacccag aggcaaggga gctgggcctt 21780 ggcatcctcg ggcttgggtgagtcacccgc agagatgcga gctccccggg cagtctggct 21840 gctggagggc cggggcacttcaaatagccc agaggccgtc atccaaagcc acaggtggag 21900 gcccgatggg gatgcccagacactcacttg aggggacatg ggcggaacct ggacagcgtc 21960 ccccacgctc acgtgtgcctttccatccac aggagaatga ctatcttcaa gactgcctcg 22020 atgccatcca gcaagacttcgtgattttta acagagaaaa gtgagtgtgt tgggggtggg 22080 tgggcgtggt ggcagagaggaggaaaatgg ggctaaggtt aaggttttct tggccacgtg 22140 agcgggcacc tgtgggcctggggtgcgtgg ccctgctctc tttggggact ctgagcagca 22200 gctatggagg ggagcggcgggaggcccctg ccaggctctg gcatgtttgt gctccacgcg 22260 gggcccgtgg ctggaatcttctggggagag acacatcatt tgcccagatg aggggtggtg 22320 acttcctagg aggccccatcagagccacgt caactccccc acccaggcac gccctcagtc 22380 tctcagcaga cctttcctgaatgtcaggcc ccaggggaca gaaagggcac agatgactgg 22440 cagcaggcaa ggcaggccagaaatagcagc agctgccacg gtggggccca agggaggatg 22500 gatgctccct ctgcccgcacggggcaagga gggcctcctg gaggaggtgg gtctgagctc 22560 ttatggacag gacgtgcagggcagcacgtg cagacggctg agggcactga ctggcaccct 22620 ggggatcaga cgaccgggtgaagaatgagg cttagccgag cctcattccc aagtcactga 22680 cctatggcac ctgcacagtcaggcctttcg gcttctggct ggaaacatgc cgagcctcgc 22740 cagcatgctc acgtgcccccacccgtcccc aggctccctg ccagtgtgtc gggagcatgg 22800 cctctccagc agacaccgagcctgtggccc acgtttgggc atccacgcca tggcctatcc 22860 catgagcccg tgggcaggtcatgggaccgt gaggccaggg aggtgggggc ataacgccct 22920 ccatgtgttc ctgccaccccaggctgaaga ggagccagga cctcccgagg gagcctctcc 22980 ccgcccctga gtccagggatgctgcggagc ctgtggagga cgaggcggaa cgctccgccc 23040 tgcccatgtg cggccccatttgcccctcgg ccgggagcgg ctaggccggg acgcccgtgg 23100 tcctcacccc ttgagctgctttggagactc ggctgccaga gggagggcca tgggccgagg 23160 cctggcattc acgttcccacccagcctggc tggcggtgcc cacagtgccc cggacacatt 23220 tcacacctca ggctcgtggtggtgcagggg acaagaggct gtgggtgcag gggacacctg 23280 tggagggcat ttcccgtgggcccccgagac ccgcctagat ggaggaagcg ctgctgggcg 23340 ccctcttacc gctcacggggagctggggcc atggatggga caggagtctt tgtccctgct 23400 cagcccggag gctgtgcacggccctcgtca caaggtgacc cttgcagcac aggccgcggg 23460 tgccccaggc tcggctcagttcttggaggt caagggcatg ggttggggta gtgggtgggg 23520 aggtgaatgt tttctagagattcaaactgc tccagcaatt tctgtatagt tttcacctct 23580 gagaattaca atgtgagaaccgctcgatgt tgcatgttct gcgtacgtcc tgtgtctgcc 23640 tggccgtcag gccggtgcctgccgtttctg gttggcctgg acttggggca gccagtgggg 23700 tgggcagctc ctcagggcagagctcccgga ccatggcttt ggggtgggtg cctgtctccg 23760 tggccctgga gccgtaaggctgtggaaggc agagacggtc ctggaggcag aggagcccag 23820 gacagcaccg tgcaccgtggagccgccgca gtgccgggca gtgcttggcc ctccataaag 23880 ggacgtatcc ctctcactgtggctgggtgg ttctgtggtt ggaactgtaa ctaactgggt 23940 aaacggcctg tgtgcttctctctggtctcg ctggaggagg acgggctcag cccgtcagcc 24000 cagcgctcca gacaggcctgtgctggtttc ctctgaggaa atgggtgtgg cgggtctgtg 24060 ccccttccca ggacagcggccatagtggac atgtgcctag acctgtgtcc atgagcccca 24120 ctgcacccct ggcaaacagggccctcccgt ccttggctgg gctgcgagat ggagatgaca 24180 acggccaaag aacatttggggaagaaccgg ccatgccacg agcagagtca gaagtccgag 24240 gggatagaat gcagcttcccgtcccccacc cacccctgtc ctctgaatca tggcagaaac 24300 tagccttcca gccctcagcagctcacatgg gggacacggc acccaaatca ccaccaggaa 24360 gggtggcccg gtctctgcgagggcccagag gcgccgtgta ctcggtggaa gtctggcgat 24420 gtcagagaca ggctcggggcaaggacaggt gtgggggttt gaataagtgc atttggggaa 24480 catggcaggg tggtgcaccttgtccttctt cgagacactg gtgaggtgtg ggtgctgtct 24540 ggttcccttg atcgccccccacactggggc agagtgggag atgctggtgt gggggacatc 24600 agctcccaca tctgggccagagggagcccc gggaaggaaa tgctgagggc ccagggcctt 24660 cgcctgggat ctgcacagctttataagcag cccagggtga gagatgggcc tgtctgtggt 24720 tcccagagac cacggcaggaaattctctgt caccatcggt gcatgggcag gggccagaga 24780 accggtggca caaggtgtccctggctctct gctcaacaaa cagcgagtgc ccagtgactg 24840 cgaccaggcc ccgctcttgggatgaggaca accgtctggg aacgtccacg caccctttat 24900 gagacacagc acgcgccagcaccgcagtca cacaccgggg gctcgggtca gcctcatagc 24960 tgcccggcct tgagtgctgggcctgcgtct gtgagcagcg cccacctggg tggcggtggt 25020 gtgtgcttca cttccacacactcccgtgca tgctccccgg ccttctgggg tggctcgggc 25080 tgtccggtga gaatgtaggcgggggggggg gggggcctct gtcccgccct ggatgttggc 25140 tgccctctgc cccgccctggatgttggctg ccctgccggc catcttccct gtgagagggt 25200 gcgctcctcc ctgccattgaggggagaaga gctgcggctg caggagtcgt cggaccagcc 25260 cacagccagc caggccccgcctgtgcagag acggcgtggg ggagaggaga cggggccttc 25320 cttccatgca caggcggcttcaaacccaga cgtctttaat gggcctgatt cacatcagag 25380 gcaggatgac tgcctgtccaggcggtgggt ggcatgcaca ggttcctggc tacagtgtcc 25440 tcagtgtaca aagctgctactaagaagcct acggaaatac acaatctgta ataagaggac 25500 agtgtcttcc taaaggatcgcaaaacttcc ctggatgagg gctacatgga agcttaggtg 25560 tgggccttgg ggtgcgtaaaagggaccctc cacgggcggg gct 25603 4 399 PRT Mus musculus 4 Val Asn Phe AspHis Phe Gln Ile Leu Arg Ala Ile Gly Lys Gly Ser 1 5 10 15 Phe Gly LysVal Cys Ile Val Gln Lys Arg Asp Thr Glu Lys Met Tyr 20 25 30 Ala Met LysTyr Met Asn Lys Gln Gln Cys Ile Glu Arg Asp Glu Val 35 40 45 Arg Asn ValPhe Arg Glu Leu Glu Ile Leu Gln Glu Ile Glu His Val 50 55 60 Phe Leu ValAsn Leu Trp Tyr Ser Phe Gln Asp Glu Glu Asp Met Phe 65 70 75 80 Met ValVal Asp Leu Leu Leu Gly Gly Asp Leu Arg Tyr His Leu Gln 85 90 95 Gln AsnVal Gln Phe Ser Glu Asp Thr Val Arg Leu Tyr Ile Cys Glu 100 105 110 MetAla Leu Ala Leu Asp Tyr Leu Arg Ser Gln His Ile Ile His Arg 115 120 125Asp Val Lys Pro Asp Asn Ile Leu Leu Asp Glu Gln Gly His Ala His 130 135140 Leu Thr Asp Phe Asn Ile Ala Thr Ile Ile Lys Asp Gly Glu Arg Ala 145150 155 160 Thr Ala Leu Ala Gly Thr Lys Pro Tyr Met Ala Pro Glu Ile PheHis 165 170 175 Ser Phe Val Asn Gly Gly Thr Gly Tyr Ser Phe Glu Val AspTrp Trp 180 185 190 Ser Val Gly Val Met Ala Tyr Glu Leu Leu Arg Gly TrpArg Pro Tyr 195 200 205 Asp Ile His Ser Ser Asn Ala Val Glu Ser Leu ValGln Leu Phe Ser 210 215 220 Thr Val Ser Val Gln Tyr Val Pro Thr Trp SerLys Glu Met Val Ala 225 230 235 240 Leu Leu Arg Lys Leu Leu Thr Val AsnPro Glu His Arg Phe Ser Ser 245 250 255 Leu Gln Asp Met Gln Thr Ala ProSer Leu Ala His Val Leu Trp Asp 260 265 270 Asp Leu Ser Glu Lys Lys ValGlu Pro Gly Phe Val Pro Asn Lys Gly 275 280 285 Arg Leu His Cys Asp ProThr Phe Glu Leu Glu Glu Met Ile Leu Glu 290 295 300 Ser Arg Pro Leu HisLys Lys Lys Lys Arg Leu Ala Lys Asn Lys Ser 305 310 315 320 Arg Asp SerSer Arg Asp Ser Ser Gln Ser Glu Asn Asp Tyr Leu Gln 325 330 335 Asp CysLeu Asp Ala Ile Gln Gln Asp Phe Val Ile Phe Asn Arg Glu 340 345 350 LysLeu Lys Arg Ser Gln Glu Leu Met Ser Glu Pro Pro Pro Gly Pro 355 360 365Glu Thr Ser Asp Met Thr Asp Ser Thr Ala Asp Ser Glu Ala Glu Pro 370 375380 Thr Ala Leu Pro Met Cys Gly Ser Ile Cys Pro Ser Ser Gly Ser 385 390395 5 368 PRT Macaca fascicularis 5 Met Tyr Ala Met Lys Tyr Met Asn LysGln Gln Cys Ile Glu Arg Asp 1 5 10 15 Glu Val Arg Asn Val Phe Arg GluLeu Gly Ile Leu Gln Glu Ile Glu 20 25 30 His Val Phe Leu Val Asn Leu TrpTyr Ser Phe Gln Asp Glu Glu Asp 35 40 45 Met Phe Met Val Val Asp Leu LeuLeu Gly Gly Asp Leu Arg Tyr His 50 55 60 Leu Gln Gln Asn Val Gln Phe SerGlu Asp Thr Val Arg Leu Tyr Ile 65 70 75 80 Cys Glu Met Ala Leu Ala LeuAsp Tyr Leu Cys Gly Gln His Ile Ile 85 90 95 His Arg Asp Val Lys Pro AspAsn Ile Leu Leu Asp Glu Arg Gly His 100 105 110 Ala His Leu Thr Asp PheAsn Ile Ala Thr Ile Ile Lys Asp Gly Glu 115 120 125 Arg Ala Thr Ala LeuAla Gly Thr Lys Pro Tyr Met Ala Pro Glu Ile 130 135 140 Phe His Ser PheVal Asn Gly Gly Thr Gly Tyr Ser Phe Glu Val Asp 145 150 155 160 Trp TrpSer Leu Gly Val Met Ala Tyr Glu Leu Leu Arg Gly Trp Arg 165 170 175 ProTyr Asp Ile His Ser Ser Asn Ala Val Glu Ser Leu Val Gln Leu 180 185 190Phe Ser Thr Val Ser Val Gln Tyr Val Pro Thr Trp Ser Arg Glu Met 195 200205 Val Ala Leu Leu Arg Lys Leu Leu Thr Val Asn Pro Glu His Arg Phe 210215 220 Ser Ser Leu Gln Asp Val Gln Ala Ala Pro Ala Leu Ala Gly Val Leu225 230 235 240 Trp Gly His Leu Ser Glu Lys Arg Val Glu Pro Asp Phe ValPro Asn 245 250 255 Lys Gly Arg Leu His Cys Asp Pro Thr Phe Glu Leu GluGlu Met Ile 260 265 270 Leu Glu Ser Arg Pro Leu His Lys Lys Lys Lys ArgLeu Ala Lys Asn 275 280 285 Lys Ser Arg Asp Asn Ser Arg Asp Ser Ser GlnSer Glu Asn Asp Tyr 290 295 300 Leu Gln Asp Cys Leu Asp Ala Ile Gln GlnAsp Phe Val Ile Phe Asn 305 310 315 320 Arg Glu Lys Leu Lys Arg Ser GlnAsp Leu Pro Ser Glu Pro Leu Pro 325 330 335 Ala Pro Glu Pro Arg Asp AlaAla Glu Pro Val Glu Asp Glu Glu Gln 340 345 350 Ser Ala Leu Pro Met CysGly Pro Ile Cys Pro Ser Ala Gly Ser Gly 355 360 365

What which is claimed is:
 1. An isolated peptide consisting of an aminoacid sequence selected from the group consisting of: (a) an amino acidsequence shown in SEQ ID NO: 2; (b) an amino acid sequence of an allelicvariant of an amino acid sequence shown in SEQ ID NO: 2, wherein saidallelic variant is encoded by a nucleic acid molecule that hybridizesunder stringent conditions to the opposite strand of a nucleic acidmolecule shown in SEQ ID NOS: 1 or 3; (c) an amino acid sequence of anortholog of an amino acid sequence shown in SEQ ID NO: 2, wherein saidortholog is encoded by a nucleic acid molecule that hybridizes understringent conditions to the opposite strand of a nucleic acid moleculeshown in SEQ ID NOS: 1 or 3; and (d) a fragment of an amino acidsequence shown in SEQ ID NO: 2, wherein said fragment comprises at least10 contiguous amino acids.
 2. An isolated peptide comprising an aminoacid sequence selected from the group consisting of: (a) an amino acidsequence shown in SEQ ID NO: 2; (b) an amino acid sequence of an allelicvariant of an amino acid sequence shown in SEQ ID NO: 2, wherein saidallelic variant is encoded by a nucleic acid molecule that hybridizesunder stringent conditions to the opposite strand of a nucleic acidmolecule shown in SEQ ID NOS: 1 or 3; (c) an amino acid sequence of anortholog of an amino acid sequence shown in SEQ ID NO: 2, wherein saidortholog is encoded by a nucleic acid molecule that hybridizes understringent conditions to the opposite strand of a nucleic acid moleculeshown in SEQ ID NOS: 1 or 3; and (d) a fragment of an amino acidsequence shown in SEQ ID NO: 2, wherein said fragment comprises at least10 contiguous amino acids.
 3. An isolated antibody that selectivelybinds to a peptide of claim
 2. 4. An isolated nucleic acid moleculeconsisting of a nucleotide sequence selected from the group consistingof: (a) a nucleotide sequence that encodes an amino acid sequence shownin SEQ ID NO: 2; (b) a nucleotide sequence that encodes of an allelicvariant of an amino acid sequence shown in SEQ ID NO: 2, wherein saidnucleotide sequence hybridizes under stringent conditions to theopposite strand of a nucleic acid molecule shown in SEQ ID NOS: 1 or3;(c) a nucleotide sequence that encodes an ortholog of an amino acidsequence shown in SEQ ID NO: 2, wherein said nucleotide sequencehybridizes under stringent conditions to the opposite strand of anucleic acid molecule shown in SEQ ID NOS: 1 or 3; (d) a nucleotidesequence that encodes a fragment of an amino acid sequence shown in SEQID NO: 2, wherein said fragment comprises at least 10 contiguous aminoacids; and (e) a nucleotide sequence that is the complement of anucleotide sequence of (a)-(d).
 5. An isolated nucleic acid moleculecomprising a nucleotide sequence selected from the group consisting of:(a) a nucleotide sequence that encodes an amino acid sequence shown inSEQ ID NO: 2; (b) a nucleotide sequence that encodes of an allelicvariant of an amino acid sequence shown in SEQ ID NO: 2, wherein saidnucleotide sequence hybridizes under stringent conditions to theopposite strand of a nucleic acid molecule shown in SEQ ID NOS: 1 or3;(c) a nucleotide sequence that encodes an ortholog of an amino acidsequence shown in SEQ ID NO: 2, wherein said nucleotide sequencehybridizes under stringent conditions to the opposite strand of anucleic acid molecule shown in SEQ ID NOS: 1 or 3; (d) a nucleotidesequence that encodes a fragment of an amino acid sequence shown in SEQID NO: 2, wherein said fragment comprises at least 10 contiguous aminoacids; and (e) a nucleotide sequence that is the complement of anucleotide sequence of (a)-(d).
 6. A gene chip comprising a nucleic acidmolecule of claim
 5. 7. A transgenic non-human animal comprising anucleic acid molecule of claim
 5. 8. A nucleic acid vector comprising anucleic acid molecule of claim
 5. 9. A host cell containing the vectorof claim
 8. 10. A method for producing any of the peptides of claim 1comprising introducing a nucleotide sequence encoding any of the aminoacid sequences in (a)-(d) into a host cell, and culturing the host cellunder conditions in which the peptides are expressed from the nucleotidesequence.
 11. A method for producing any of the peptides of claim 2comprising introducing a nucleotide sequence encoding any of the aminoacid sequences in (a)-(d) into a host cell, and culturing the host cellunder conditions in which the peptides are expressed from the nucleotidesequence.
 12. A method for detecting the presence of any of the peptidesof claim 2 in a sample, said method comprising contacting said samplewith a detection agent that specifically allows detection of thepresence of the peptide in the sample and then detecting the presence ofthe peptide.
 13. A method for detecting the presence of a nucleic acidmolecule of claim 5 in a sample, said method comprising contacting thesample with an oligonucleotide that hybridizes to said nucleic acidmolecule under stringent conditions and determining whether theoligonucleotide binds to said nucleic acid molecule in the sample.
 14. Amethod for identifying a modulator of a peptide of claim 2, said methodcomprising contacting said peptide with an agent and determining if saidagent has modulated the function or activity of said peptide.
 15. Themethod of claim 14, wherein said agent is administered to a host cellcomprising an expression vector that expresses said peptide.
 16. Amethod for identifying an agent that binds to any of the peptides ofclaim 2, said method comprising contacting the peptide with an agent andassaying the contacted mixture to determine whether a complex is formedwith the agent bound to the peptide.
 17. A pharmaceutical compositioncomprising an agent identified by the method of claim 16 and apharmaceutically acceptable carrier therefor.
 18. A method for treatinga disease or condition mediated by a human kinase protein, said methodcomprising administering to a patient a pharmaceutically effectiveamount of an agent identified by the method of claim
 16. 19. A methodfor identifying a modulator of the expression of a peptide of claim 2,said method comprising contacting a cell expressing said peptide with anagent, and determining if said agent has modulated the expression ofsaid peptide.
 20. An isolated human kinase peptide having an amino acidsequence that shares at least 70% homology with an amino acid sequenceshown in SEQ ID NO:
 2. 21. A peptide according to claim 20 that sharesat least 90 percent homology with an amino acid sequence shown in SEQ IDNO:
 2. 22. An isolated nucleic acid molecule encoding a human kinasepeptide, said nucleic acid molecule sharing at least 80 percent homologywith a nucleic acid molecule shown in SEQ ID NOS: 1 or
 3. 23. A nucleicacid molecule according to claim 22 that shares at least 90 percenthomology with a nucleic acid molecule shown in SEQ ID NOS: 1 or 3.